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WO2003010197A2 - Polynucleotides et polypeptides gmg-1 et leurs utilisations - Google Patents

Polynucleotides et polypeptides gmg-1 et leurs utilisations Download PDF

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Publication number
WO2003010197A2
WO2003010197A2 PCT/IB2002/003402 IB0203402W WO03010197A2 WO 2003010197 A2 WO2003010197 A2 WO 2003010197A2 IB 0203402 W IB0203402 W IB 0203402W WO 03010197 A2 WO03010197 A2 WO 03010197A2
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Prior art keywords
polypeptide
ggmg
gmg
sequence
fragment
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PCT/IB2002/003402
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English (en)
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WO2003010197A3 (fr
Inventor
John Lucas
Aaron Scalia
Deno Dialynas
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Genset S.A.
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Application filed by Genset S.A. filed Critical Genset S.A.
Priority to AU2002330672A priority Critical patent/AU2002330672A1/en
Publication of WO2003010197A2 publication Critical patent/WO2003010197A2/fr
Publication of WO2003010197A3 publication Critical patent/WO2003010197A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—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
    • 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 present invention relates to the field of metabolic research, in particular the discovery of compounds effective for treating obesity and obesity-related diseases and disorders.
  • the obesity-related diseases or disorders envisioned to be treated by the methods of the invention include, but are not limited to, hyperlipidemia, atherosclerosis, diabetes, and hypertension.
  • Obesity is a public health problem that is serious, widespread, and increasing. In the United States, 20 percent of the population is obese; in Europe, a slightly lower percentage is obese [Friedman (2000) Nature 404:632-634]. Obesity is associated with increased risk of hypertension, cardiovascular disease, diabetes, and cancer as well as respiratory complications and osteoarthritis [Kopelman (2000) Nature 404:635-643]. Even modest weight loss ameliorates these associated conditions.
  • leptin ob and its receptor (db)
  • pro-opiomelanocortin Pome
  • melanocortin-4-receptor Mc4r
  • agouti protein A
  • carboxypeptidase E fat
  • 5-hydroxytryptamine receptor 2C Htr2c
  • Nhlh2 nescient basic helix-loop-helix 2
  • PCSK1 prohormone convertase 1
  • tubby protein tubby [rev'd in Barsh et al. (2000) Nature 404:644-651].
  • the instant invention is based on the discovery that fragments of the full-length GMG-1 polypeptide comprising the globular domain, termed gGMG-1 polypeptide fragments, form homotrimers having unexpected effects in vitro and in vivo, including utility for weight reduction, prevention of weight gain, and control of blood glucose levels in humans and other mammals.
  • the invention is further based on the discovery that multimers of gGMG-1 homotrimer formed through disulfide linkage at the cysteine residue within the N-terminally disposed unique region have lower specific activity for the activities disclosed herein than does non-multimeric gGMG-1 homotrimer.
  • the instant invention is yet further based on the discovery that gGMG-1 polypeptide fragments comprising all or part of the collagen-like region form more stable gGMG-1 homotrimers having the activities disclosed herein.
  • non-multimeric gGMG-1 homotrimer of the invention is radically more effective and thus can be provided at levels that are feasible for treatments in humans.
  • the invention is drawn to gGMG-1 polypeptide fragments, polynucleotides encoding said gGMG-1 polypeptide fragments, vectors comprising said gGMG-1 polynucleotides, and cells recombinant for said gGMG-1 polynucleotides, as well as to pharmaceutical and physiologically acceptable compositions comprising said gGMG-1 polypeptide fragments and methods of administering said gGMG-1 pharmaceutical and physiologically acceptable compositions in order to reduce body weight or to treat obesity-related diseases and disorders, wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region, and further wherein said gGMG-1 polypeptide fragment does not comprise the cysteine residue within the N-terminally disposed unique region either because said cysteine residue has been substituted with an amino acid other than cysteine or because said fragment does not span said cysteine residue.
  • Assays for identifying agonists and antagonists of obesity-related activity are also part of the invention.
  • Antagonists of homotrimeric gGMG-1 polypeptide fragment activity should be effective in the treatment of other metabolic-related diseases or disorders of the invention including cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.
  • said individual is a mammal, preferably a human.
  • the invention features a purified, isolated, or recombinant gGMG-1 polypeptide fragment, wherein said gGMG-1 polypeptide fragment forms homotrimers having unexpected activity, wherein unexpected said activity is selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, wherein said gGMG-1 polypeptide comprises all or part of the collagen-like region, and wherein said gGMG-1 polypeptide fragment comprises a substitution of an amino acid other than cysteine for the cysteine within the N- terminally disposed unique region selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucme, methionine, asparagines, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine, preferably wherein said substituted amino acid is serine.
  • said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 and not more than 285 consecutive amino acids of SEQ ID NO:2 wherein said polypeptide fragment comprises all or part of the collagen-like region, and wherein the cysteine at position 36 is replaced by said substitute amino acid; or at least 6 and not more than 217 consecutive amino acids of SEQ ID NO:4 wherein said polypeptide comprises all or part of the collagen-like region and wherein the cysteine at position 36 is replaced by said substitute amino acid, hi other preferred embodiments, gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 4
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 16-217, 17-217, 18-217, 19-217, 20-217, 21-217, 22-217, 23-217, 24-217, 25-217, 26-217, 27-217, 28-217, 29-217, 30-217, 31-217, 32-217, 33-217, 34-217, 35-217, 36-217, 37-217, 38-217, 39-217, 40-217, 41-217, 42-217, 43-217, 44-217, 16-217, 17-217, 18-217, 19-217, 20-217, 21-217, 22-217, 23-217, 24-217, 25-217, 26-217, 27-217, 28-217, 29-217, 30-217, 31-217, 32-217, 33-217, 34-217, 35-217, 36-217, 37-217, 38-217, 39-217, 40-217, 41-217, 42-217, 43-217, 44-217, 16-217,
  • gGMG-1 polypeptide fragments having unexpected activity are human.
  • said polypeptide fragment comprises an amino acid sequence at least
  • the invention features an GMG-1 polypeptide fragment wherein said GMG-1 polypeptide fragment forms homotrimers having unexpected activity selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, wherein said GMG-1 polypeptide fragment comprises, consists essentially of, or consists of a purified, isolated, or recombinant gGMG-1 polypeptide fragment, wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region, and wherein said gGMG-1 polypeptide fragment comprises a substitution of an amino acid other than cysteine for the cysteine within the N-terminally disposed unique region selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagines, proline, glutamine, arginine, serine, threonine, valine, trypto
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 16-285, 17-285,
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 16-217, 17-217, 18-217, 19-217, 20-217, 21-217, 22-217, 23-217, 24-217, 25-217, 26-217, 27-217, 28-217, 29-217, 30-217, 31-217, 32-217, 33-217, 34-217, 35-217, 36-217, 37-217, 38-217, 39-217, 40-217,
  • the invention features a purified, isolated, or recombinant gGMG-1 polypeptide fragment, wherein said gGMG-1 polypeptide fragment forms homotrimers having unexpected activity, wherein unexpected said activity is selected from the group consisting of prevention of weight gain, weight reduction, and maintenance of weight loss, wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region, and wherein said gGMG-1 polypeptide fragment comprises a substitution of an amino acid other than cysteine for the cysteine within the N-terminally disposed unique region selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagines, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine, preferably wherein said substituted amino acid is serine.
  • said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 and not more than 285 consecutive amino acids of SEQ ID NO:2 wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region and wherein the cysteine at position 36 is replaced by said substitute amino acid or at least 6 and not more than 217 consecutive amino acids of SEQ ID NO:4 wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region and wherein the cysteine at position 36 is replaced by said substitute amino acid.
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-285, 71-285, 72-285,
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 16-217, 17-217, 18-217, 19- 217, 20-217, 21-217, 22-217, 23-217, 24-217, 25-217, 26-217, 27-217, 28-217, 29-217, 30-217, 31- 217, 32-217, 33-217, 34-217, 35-217, 36-217, 37-217, 38-217, 39-217, 40-217, 41-217, 42-217, 43- 217, 44-217, 45-217, 46-217, 47-217, 48-217, 49-217, 50-217, 51-217, 52-217, 53-217, 54-217, 55- 217, 56-217, 57-217, 58-217, 59-217, 60-217, 61-217, 62-217, 63-217, 64-217, 65-217, 66-217, 67- 217, 68-217, 69-217, 70-217,
  • gGMG-1 polypeptide fragment having unexpected activity is human.
  • said polypeptide fragment comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of SEQ ID NO:2 or SEQ ID NO:4. Any gGMG-1 polypeptide fragment that forms homotrimers with activity, as described above, may be excluded.
  • the invention features an GMG-1 polypeptide fragment wherein said GMG-1 polypeptide fragment forms homotrimers having unexpected activity selected from the group consisting of prevention of weight gain, weight reduction, and maintenance of weight loss and wherein said polypeptide fragment comprises, consists essentially of, or consists of a purified, isolated, or recombinant gGMG-1 polypeptide fragment, wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region, and wherein said gGMG-1 polypeptide fragment comprises a substitution of an amino acid other than cysteine for the cysteine within the N-terminally disposed unique region selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagines, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and
  • said gGMG-1 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 16 to 285 of SEQ ID NO:2 wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region and wherein the cysteine at position 36 is replaced by said substitute amino acid or at least 6 consecutive amino acids of amino acids 16 to 217 of SEQ ID NO:4 wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region wherein the cysteine at position 36 is replaced by said substitute amino acid.
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25- 285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37- 285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 16-217, 17-217, 18-217, 19-217, 20-217, 21-217, 22-217, 23-217, 24-217, 25-217, 26-217, 27-217, 28-217, 29-217, 30-217, 31-217, 32-217, 33-217, 34-217, 35-217, 36-217, 37-217, 38-217, 39-217, 40-217, 41-217, 42-217, 43-217, 44-217, 16-217, 17-217, 18-217, 19-217, 20-217, 21-217, 22-217, 23-217, 24-217, 25-217, 26-217, 27-217, 28-217, 29-217, 30-217, 31-217, 31-217, 3
  • said gGMG-1 fragment comprises, consists essentially of, or consists of, an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 16-285 of SEQ ID NO:2 or at least 75% identical to amino acids 16-217 of SEQ ID NO:4.
  • Any gGMG-1 polypeptide fragment that forms homotrimers with activity, as described above, may be excluded.
  • said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 and not more than 285 consecutive amino acids of SEQ ID NO:2 or at least 6 and not more than 217 consecutive amino acids of SEQ ID NO:4.
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 45-285, 47-285, 126-285, 127-285, 132-285, 133-285, or 134-285 of SEQ ID NO:2.
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 45-217, 47-217, or 88-217 of SEQ ID NO:4.
  • gGMG-1 polypeptide fragment having unexpected activity is human.
  • said polypeptide fragment comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94% > , 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of
  • SEQ ID NO:2 SEQ ID NO:4. Any gGMG-1 polypeptide fragment that forms homotrimers with activity, as described above, may be excluded.
  • the invention features an GMG-1 polypeptide fragment wherein said GMG-1 polypeptide fragment forms homotrimers having unexpected activity selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity, wherein said GMG-1 polypeptide fragment comprises, consists essentially of, or consists of a purified, isolated, or recombinant gGMG-1 polypeptide fragment, wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region and wherein said gGMG-1 polypeptide fragment does not span the cysteine residue within the N-terminally disposed unique region.
  • said gGMG-1 polypeptide fragment comprises, consists essentially of, or consists of, an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 40-285 of SEQ ID NO:2 or at least 75% identical to amino acids 40-217 of SEQ ID NO:4. Any gGMG-1 polypeptide fragment that forms homotrimers with activity, as described above, may be excluded.
  • said polypeptide fragment comprises, consists essentially of, or consists of, at least 6 and not more than 285 consecutive amino acids of SEQ ID NO:2 or at least 6 and not more than 217 consecutive amino acids of SEQ ID NO:4.
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 45-285, 47-285, 126-285, 127-285, 132-285, 133-285, or 134-285 of SEQ ID NO:
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 45-217, 47-217, or 88-217 of SEQ ID NO:4. In most preferred embodiments, gGMG-1 polypeptide fragment having unexpected activity is human. In other further preferred embodiments, said polypeptide fragment comprises an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding consecutive amino acids of SEQ ID NO:2 or SEQ ID NO:4. Any gGMG-1 polypeptide fragment that forms homotrimers with activity, as described above, may be excluded.
  • said gGMG-1 polypeptide fragment comprises, consists essentially of, or consists of, at least 6 consecutive amino acids of amino acids 40-285 of SEQ ID NO:2 comprising all or part of the collagen-like region or at least 6 consecutive amino acids of amino acids 40-217 of SEQ ID NO:4 comprising all or part of the collagen-like region.
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 45-285, 47-285, 126-285, 127-285, 132-285, 133-285, or 134-285 of SEQ ID NO:2.
  • gGMG-1 polypeptide fragments having unexpected activity are selected from amino acids 45-217, 47-217, or 88-217 of SEQ ID NO:4. In most preferred embodiments, gGMG-1 polypeptide fragment having unexpected activity is human. Alternatively, said gGMG-1 fragment comprises, consists essentially of, or consists of, an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding amino acids 40- 285 of SEQ ID NO:2 or at least 75% identical to amino acids 40-217 of SEQ ID NO:4.
  • any gGMG- 1 polypeptide fragment that forms homotrimers with activity may be excluded.
  • the invention features a purified, isolated, or recombinant gGMG-1 polypeptide fragment, wherein said gGMG-1 polypeptide fragment forms homotrimers having significantly greater activity than a monomeric gGMG-1 polypeptide fragment, wherein unexpected said activity is selected from the group consisting of inhibiting smooth muscle proliferation, inhibiting expression of proinflammatory cytokines, inhibiting expression of cell adhesion molecules, and inhibiting expression of tissue factor, wherein said gGMG-1 polypeptide fragment comprises all or part of the collagen-like region, and wherein said gGMG-1 polypeptide fragment comprises a substitution of an amino acid other than cysteine for the cysteine within the N- terminally disposed unique region selected from the group consisting of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine
  • the gGMG-1 polypeptide fragment forms homotrimers able to lower circulating (either blood, serum or plasma) levels (concentration) of: (i) free fatty acids, (ii) glucose, and/or (iii) triglycerides.
  • Further preferred gGMG-1 polypeptide fragments form homotrimers that demonstrate free fatty acid level lowering activity, glucose level lowering activity, and/or triglyceride level lowering activity, have an activity that is significantly greater than full- length GMG-1 at the same molar concentration, have a greater than transient activity and/or have a sustained activity.
  • gGMG-1 polypeptide fragments are those that form homotrimers that maintain weight loss, preferably in individuals who were previously "obese” and are now “healthy” (as defined herein) .
  • gGMG-1 polypeptide fragments are those that form homotrimers that significantly stimulate muscle lipid or free fatty acid oxidation as compared to full-length GMG-1 polypeptides at the same molar concentration. Further preferred gGMG-1 polypeptide fragments are those that form homotrimers that cause C2C12 cells differentiated in the presence of said fragments to undergo at least 10%, 20%, 30%, 35%, or 40% more oleate oxidation as compared to untreated cells or cells treated with full-length GMG-1.
  • gGMG-1 polypeptide fragments are those that form homotrimers that are at least 30% more efficient than full-length GMG-1 at increasing leptin uptake in a liver cell line (preferably BPRCL mouse liver cells (ATCC CRL-2217)). Further preferred gGMG-1 polypeptide fragments are those that form homotrimers that significantly reduce the postprandial increase in plasma free fatty acids, particularly following a high fat meal.
  • gGMG-1 polypeptide fragments are those that form homotrimers that significantly reduce or eliminate ketone body production, particularly following a high fat meal. Further preferred gGMG-1 polypeptide fragments are those that form homotrimers that increase glucose uptake in skeletal muscle cells.
  • gGMG-1 polypeptide fragments are those that form homotrimers that increase glucose uptake in neuronal cells.
  • gGMG-1 polypeptide fragments are those that form homotrimers that improve insulin sensitivity.
  • gGMG-1 polypeptide fragments are those that form homotrimers that inhibit the progression from impaired glucose tolerance to insulin resistance.
  • gGMG-1 polypeptide fragments are those that form homotrimers that increase muscle mass, preferably those that increase muscle cell number, more preferably those that increase muscle fiber number.
  • gGMG-1 polypeptide fragments are those that form homotrimers that promote an increase in body girth, preferably fragments that promote an increase in muscle mass. Further preferred gGMG-1 polypeptide fragments promote growth rate, preferably promoting an increase in growth rate greater than an average growth rate in the absence of gGMG-1 polypeptide fragments.
  • gGMG-1 polypeptide fragments are those that form homotrimers that promote growth rate in newborn mammals, preferably cow, goat, sheep, rabbit, mouse, rat, pig, dog, or human newborns, more preferably human newborns between the ages of 0-6 months of age, most preferably human newborn between the ages of 0-3 months.
  • Further preferred gGMG-1 polypeptide fragments are those that form homotrimers that promote growth rate in newborn underweight or premature mammals, preferably cow, goat, sheep, rabbit, mouse, rat, pig, dog, or human underweight or premature newborns, more preferably human underweight or premature newborns between the ages of 0-6 months of age, most preferably human underweight or premature newborns between the ages of 0-3 months of age.
  • gGMG-1 polypeptide fragments are those that form homotrimers in vitro and/or in vivo. More preferred gGMG-1 polypeptide fragments are those that form homotrimers in vitro and/or in vivo, wherein at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of said gGMG-1 polypeptide fragment comprises said homotrimer.
  • gGMG-1 polypeptide fragments are those that form homotrimers in vitro and/or in vivo having activity selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity. Also most particularly preferred gGMG-1 polypeptide fragments are those that form homotrimers in vitro and/or in vivo having activity selected from the group consisting of prevention of weight gain, weight reduction, and maintenance of weight loss. Any gGMG-1 polypeptide fragment that forms homotrimers with activity, as described above, may be excluded.
  • heterologous polypeptides comprising a gGMG-1 polypeptide fragment of the invention. More preferred is said heterologous polypeptide comprised of a signal peptide N-terminally fused to said gGMG-1 polypeptide of the invention.
  • said signal peptide is human zinc-alpha 2-glycoprotein signal peptide of amino acid sequence MVRMVPVLLSLLLLLGPAVP, preferably encoded by the polynucleotide of sequence atggtaagaatggtgcctgtcctgctgtctctgctgggtcctgctgtcccccc.
  • the invention features a purified, isolated, or recombinant polynucleotide encoding said gGMG-1 polypeptide fragment or full-length GMG-1 polypeptide described in the first aspect, or the complement thereof.
  • the polynucleotides are DNA, RNA,
  • DNA/RNA hybrids single-stranded, and double-stranded.
  • the invention features a recombinant vector comprising, consisting essentially of, or consisting of, said polynucleotide described in the second aspect.
  • the invention features a recombinant cell comprising, consisting essentially of, or consisting of, said recombinant vector described in the third aspect.
  • Preferred said recombinant cell is prokaryotic or eukaryotic recombinant cell.
  • Preferred said prokaryotic recombinant cell is E. coli recombinant cell.
  • Preferred said eukaryotic recombinant cell is mammalian recombinant cell.
  • Particularly preferred mammalian recombinant cell is selected from the group consisting of COS recombinant cell, Chinese hamster ovary (CHO) recombinant cell, human embryonic kidney (HEK) recombinant cell, and 3T3-L1 adipocyte recombinant cell.
  • a further embodiment includes a host cell recombinant for a polynucleotide of the invention.
  • Preferred said host cell is prokaryotic or eukaryotic host cell.
  • Preferred said prokaryotic host cell is E. coli host cell.
  • Preferred said eukaryotic host cell is mammalian host cell.
  • the invention features a pharmaceutical or physiologically acceptable composition comprising, consisting essentially of, or consisting of, said gGMG-1 polypeptide fragment described in the first aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent. More preferred said pharmaceutical or physiologically acceptable composition comprises, consists essentially of, or consists of homotrimer of said gGMG-1 polypeptide fragment described in the first aspect and, alternatively, a pharmaceutical or physiologically acceptable diluent.
  • said pharmaceutical or physiologically acceptable composition preferably at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of said gGMG-1 polypeptide fragment of the first aspect comprises homotrimer.
  • the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass said pharmaceutical or physiologically acceptable composition described in the fifth aspect.
  • a method of reducing body fat mass comprising providing or administering to individuals in need thereof said pharmaceutical or physiologically acceptable composition described in the fifth aspect.
  • a method of increasing lean body mass comprising providing or administering to individuals in need thereof said pharmaceutical or physiologically acceptable composition described in the fifth aspect.
  • a method of increasing the growth rate of body girth or length comprising providing or administering to individuals in need thereof said pharmaceutical or physiologically acceptable composition described in the fifth aspect.
  • the present invention may be used in complementary therapy of obese patients to improve their weight in combination with a weight reducing agent.
  • the invention features a method of maintaining a reduced body mass comprising providing or administering to individuals in need of maintaining a reduced body mass said pharmaceutical or physiologically acceptable composition described in the fifth aspect. Further preferred is a method of maintaining a reduced body fat mass that comprises, providing or administering to individuals in need thereof said pharmaceutical or physiologically acceptable composition described in the fifth aspect, returning energy intake to a normal level in said individual, and maintaining increased energy expenditure in said individual.
  • said individual is able to maintain a stable weight that is 10-20% below their obese weight (as described herein).
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used in combination with reduced energy intake and/or increased energy expenditure as a method of maintaining weight loss.
  • the identification of said individuals in need of reducing body mass to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-1 single nucleotide polymorphisms (SNPs) or measuring GMG-1 polypeptide or mRNA levels in clinical samples from said individuals.
  • said clinical samples are selected from the group consisting of plasma, urine, and saliva.
  • a gGMG-1 polypeptide fragment of the present invention is administered to an individual with at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in blood, serum or plasma levels of full-length GMG-1 or the naturally proteolytically cleaved GMG-1 fragment as compared to healthy, non-obese patients.
  • the invention features a method of preventing or treating an obesity- related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.
  • the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-1 single nucleotide polymorphisms (SNPs) or measuring GMG-1 polypeptide or mRNA levels in clinical samples from said individuals.
  • said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke,
  • Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.
  • Yet other metabolic-related diseases or disorders of the invention including cachexia, wasting, AIDS-related weight loss, cancer- related weight loss, anorexia, and bulimia, preferred embodiments, said individual is a mammal, preferably a human.
  • the invention features a method of preventing or treating an inflammation-related disease or disorder comprising providing or administering to an individual in need of such treatment said pharmaceutical or physiologically acceptable composition described in the fifth aspect.
  • the identification of said individuals in need of such treatment to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-1 single nucleotide polymorphisms (SNPs) or measuring GMG-1 polypeptide or mRNA levels in clinical samples from said individuals.
  • SNPs single nucleotide polymorphisms
  • said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • said inflammation-related disease or disorder is selected from the group consisting vascular disorders, diseases and injuries, particularly those caused by excessive inflammatory responses, such as atherosclerosis, angina pectoris, myocardial infarction, deep vein thrombosis, peripheral arterial occlusion, coronary heart disease (CDH), coronary artery disease (CAD), heart failure, transient ischemic attack, post-angioplasty restenoses, myelopoiesis, and disseminated intravascular coagulation (DIC).
  • said individual is a mammal, preferably a human.
  • embodiments of the present invention includes methods of causing or inducing a desired biological response in an individual comprising the steps of: providing or administering to an individual a composition comprising a polypeptide of the first aspect, wherein said biological response is selected from the group consisting of:
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.
  • IDDM Insulin Dependent Diabetes Mellitus
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type ⁇ diabetes) in combination with insulin therapy.
  • NIDDM Noninsulin Dependent Diabetes Mellitus
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control blood glucose in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) alone, without combination of insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to control body weight in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) alone, without combination of insulin therapy.
  • the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.
  • the present invention further provides a method of improving the body weight or glucose control of NIDDM patients alone, without an oral insulin secretagogue or an insulin sensitising agent.
  • the present invention may be used in complementary therapy of IDDM patients to improve their weight or glucose control in combination with an oral insulin secretagogue or an insulin sensitising agent.
  • the oral insulin secretagogue is 1,1- dimethyl-2-(2-morpholino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.
  • BTS67582 1,1- dimethyl-2-(2-morpholino phenyl)guanidine fumarate
  • a sulphonylurea selected from tolbutamide, tolazamide, chlorpropamide, glibenclamide, glimepiride, glipizide and glidazide.
  • the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.
  • the present invention further provides a method of improving the body weight or glucose control of IDDM patients alone, without an oral insulin secretagogue or an insulin sensitising agent, i a further preferred embodiment, the present invention may be administered either concomitantly or concurrently, with the oral insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially (either before or after the secretagogue or either before or after the sensitising agent).
  • the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an oral insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose control in NIDDM or IDDM patients.
  • the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Noninsulin Dependent Diabetes Mellitus (NIDDM, Type II diabetes) in combination with insulin therapy.
  • NIDDM Noninsulin Dependent Diabetes Mellitus
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some persons with Insulin Dependent Diabetes Mellitus (IDDM, Type I diabetes) in combination with insulin therapy.
  • IDDM Insulin Dependent Diabetes Mellitus
  • the invention features a method of making the gGMG-1 polypeptide fragment described in the first aspect, wherein said method is selected from the group consisting of: proteolytic cleavage, recombinant methodology and artificial synthesis.
  • the present invention provides a method of making a recombinant gGMG-
  • the method comprising providing a transgenic, non-human mammal whose milk contains said recombinant gGMG-1 polypeptide fragment or full-length protein, and purifying said recombinant gGMG-1 polypeptide fragment or said full-length GMG-1 polypeptide from the milk of said non-human mammal.
  • said non-human mammal is a cow, goat, sheep, rabbit, or mouse.
  • the method comprises purifying a recombinant mature GMG-1 polypeptide absent the signal peptide from said milk, and further comprises cleaving said protein in vitro to obtain a desired gGMG-1 polypeptide fragment.
  • the invention features a use of the polypeptide described in the first aspect for freatment of obesity-related diseases and disorders and/or reducing body mass.
  • said obesity-related diseases and disorders are selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (TDDM or Type I diabetes).
  • Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • the invention further features a use of the polypeptide of the first aspect for prevention of weight gain, for weight reduction, and or for maintenance of weight loss.
  • said individual is a mammal, preferably a human.
  • the invention features a use of the polypeptide described in the first aspect for the preparation of a medicament for the freatment of obesity-related diseases and disorders and/or for reducing body mass.
  • said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent
  • Diabetes Mellitus or Type II diabetes
  • IDDM Insulin Dependent Diabetes Mellitus
  • Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.
  • Yet other metabolic-related diseases or disorders of the invention including cachexia, wasting, AJDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.
  • said individual is a mammal, preferably a human.
  • the invention further features a use of the polypeptide of the first aspect for the preparation of a medicament for prevention of weight gain, for weight reduction, and/or for maintenance of weight loss.
  • said individual is a mammal, preferably a human.
  • the invention provides a polypeptide of the first aspect of the invention, or a composition of the fifth aspect of the invention, for use in a method of treatment of the human or animal body.
  • the invention features methods of reducing body weight comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or the polypeptide described in the first aspect. Where the reduction of body weight is practiced for cosmetic purposes, the individual has a BMI of at least 20 and no more than 25.
  • the individual may have a BMI of at least 20.
  • One embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 25.
  • Another embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 30.
  • Yet another embodiment provides for the freatment of individuals with BMI values of at least 40.
  • the BMI value should be at least 15 and no more than 20.
  • the invention features methods of maintaining weight loss comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or the polypeptide described in the first aspect. Where the maintenance of weight loss is practiced for cosmetic purposes, the individual has a BMI of at least 20 and no more than 25.
  • the individual may have a BMI of at least 20.
  • One embodiment for the freatment of obesity by means of maintaining weight loss provides for the treatment of individuals with BMI values of at least 25.
  • Another embodiment for the freatment of obesity by means of maintaining weight loss provides for the freatment of individuals with BMI values of at least 30.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body mass and/or for treatment or prevention of obesity-related diseases or disorders.
  • said obesity-related disease or disorder is selected from the group consisting of obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NTDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).
  • Diabetes-related complications to be freated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.
  • Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.
  • said individual is a mammal, preferably a human.
  • the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping GMG-1 single nucleotide polymorphisms (SNPs) or measuring GMG-1 polypeptide or mRNA levels in clinical samples from said individuals.
  • SNPs single nucleotide polymorphisms
  • said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing an inflammatory response, preferrably an inflammatory response related to a vascular dirorder or injury, and/or for treatment or prevention of inflammation-related diseases or disorders.
  • said inflammation-related disease or disorder is selected from the group consisting of vascular disorders, diseases and injuries, particularly those caused by excessive inflammatory responses, such as atherosclerosis, angina pectoris, myocardial infarction, deep vein thrombosis, peripheral arterial occlusion, coronary heart disease (CDH), coronary artery disease (CAD), heart failure, transient ischemic attack, post-angioplasty restenoses, myelopoiesis, and disseminated intravascular coagulation (DIC).
  • said individual is a mammal, preferably a human.
  • the identification of said individuals to be treated with said pharmaceutical or physiologically acceptable composition comprises genotyping OBG3 single nucleotide polymorphisms (SNPs) or measuring OBG3 polypeptide or mRNA levels in clinical samples from said individuals.
  • SNPs single nucleotide polymorphisms
  • said clinical samples are selected from the group consisting of blood, serum, plasma, urine, and saliva.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for reducing body weight for cosmetic reasons.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect for maintaining weight loss for cosmetic reasons.
  • the invention features methods of treating insulin resistance comprising providing to an individual said pharmaceutical or physiologically acceptable composition described in the fifth aspect, or the polypeptide described in the first aspect.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with normal glucose tolerance (NGT) who are obese or who have fasting hyperinsulinemia, or who have both.
  • NTT normal glucose tolerance
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with gestational diabetes.
  • Gestational diabetes refers to the development of diabetes in an individual during pregnancy, usually during the second or third trimester of pregnancy.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating individuals with impaired fasting glucose (IFG).
  • Impaired fasting glucose (IFG) is that condition in which fasting plasma glucose levels in an individual are elevated but not diagnostic of overt diabetes, i.e. plasma glucose levels of less than 126 mg/dl and greater than or equal to 110 mg/dl.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating impaired glucose tolerance (IGT) in an individual.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of preventing IGT in an individual.
  • IGT impaired glucose tolerance
  • the invention provides methods for reducing and/or preventing the appearance of Insulin-Resistance Syndrome.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having polycystic ovary syndrome (PCOS).
  • PCOS polycystic ovary syndrome
  • the invention provides methods for reducing insulin resistance, normalizing blood glucose thus treating and/or preventing PCOS.
  • the invention features the pharmaceutical or physiologically acceptable composition described in the fifth aspect in a method of treating a subject having insulin resistance.
  • a subject having insulin resistance is treated according to the methods of the invention to reduce or cure the insulin-resistance.
  • the methods of the invention may prevent or reduce infections and cancer.
  • the methods of the invention are used to prevent the development of insulin resistance in a subject, e.g., those known to have an increased risk of developing insulin-resistance.
  • the invention features a method of using homotrimeric gGMG-1 polypeptide fragment in a method of screening compounds for one or more antagonists of homotrimeric gGMG-1 polypeptide fragment activity, wherein said activity is selected from but not restricted to weight reduction, maintenance of weight loss, lipid partitioning, lipid metabolism, and insulin-like activity.
  • said compound is selected from but is not restricted to small molecular weight organic or inorganic compound, protein, peptide, carbohydrate, or lipid.
  • compositions of the invention may further comprise any combination of gGMG-1 polypeptide fragment of the first aspect, insulin, insulin secretagogues or insulin sensitising agents such that the composition produces a biological effect greater than the expected effect for said gGMG-1 polypeptide fragment administered alone rather than in combination with insulin, insulin secretagogues or insulin sensitising agents.
  • said biological function includes, but is not limited to, free fatty acid level lowering activity, glucose level lowering activity, triglyceride level lowering activity, stimulating adipose lipolysis, stimulating muscle lipid or free fatty acid oxidation, increasing leptin uptake in a liver cell line, significantly reducing the postprandial increase in plasma free fatty acids or glucose due to a high fat meal, significantly reducing or eliminate ketone body production as the result of a high fat meal, increasing glucose uptake in skeletal muscle cells, adipose cells, red blood cells or the brain, increasing insulin sensitivity, inhibiting the progression from impaired glucose tolerance to insulin resistance, reducing body mass, decreasing fat mass, increasing lean muscle mass, preventing or treating an metabolic-related disease or disorder, controlling blood glucose in some persons with Noninsulin Dependent Diabetes Mellitus or Noninsulin Dependent Diabetes Mellitus, treating insulin resistance or preventing the development of insulin resistance.
  • SEQ ID NO: 1 is the nucleotide sequence of cDNA with an open reading frame which location is indicated as featured.
  • SEQ ID NO: 2 is the amino acid sequence of protein encoded by the cDNA of SEQ ID NO:l.
  • SEQ ID NO: 3 is the nucleotide sequence of cDNA with an open reading frame which location is indicated as featured.
  • SEQ ID NO:4 is the amino acid sequence of protein encoded by the cDNA of SEQ ID NO:3.
  • purified is used herein to describe a polynucleotide or polynucleotide vector of the invention that has been separated from other compounds including, but not limited to, other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide). Purified can also refer to the separation of covalently closed polynucleotides from linear polynucleotides, or vice versa, for example.
  • a polynucleotide is substantially pure when at least about 50%, 60%, 75%, or 90% of a sample contains a single polynucleotide sequence. In some cases this involves a determination between conformations (linear versus covalently closed).
  • a substantially pure polynucleotide typically comprises about 50, 60, 70, 80, 90, 95, 99% weight/weight of a nucleic acid sample.
  • Polynucleotide purity or homogeneity may be indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel. For certain purposes, higher resolution can be achieved by using HPLC or other means well known in the art.
  • purified is used herein to describe a polypeptide of the invention that has been separated from other compounds including, but not limited to, nucleic acids, lipids, carbohydrates and other proteins.
  • a polypeptide is substantially pure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the polypeptide molecules of a sample have a single amino acid sequencesequence.
  • a substantially pure polypeptide typically comprises about 50%, 60%, 70%, 80%,
  • polypeptide purity or homogeneity is indicated by a number of methods well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polypeptide band upon staining the gel. For certain purposes, higher resolution can be achieved by using HPLC or other methods well known in the art.
  • purified does not require absolute purity; rather, it is intended as a relative definition. Purification of starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. Alternatively, purification may be expressed as "at least" a percent purity relative to heterologous polynucleotides (DNA, RNA or both) or polypeptides.
  • the polynucleotides or polypeptides of the present invention are at least; 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 98%, 99%, 99.5% or 100% pure relative to heterologous polynucleotides or polypeptides.
  • the polynucleotides or polypeptides have an "at least" purity ranging from any number, to the thousandth position, between 90% and 100% (e.g., at least 99.995% pure) relative to heterologous polynucleotides or polypeptides. Additionally, purity of the polynucleotides or polypeptides may be expressed as a percentage (as described above) relative to all materials and compounds other than the carrier solution. Each number, to the thousandth position, may be claimed as individual species of purity.
  • isolated requires that the material be removed from its original environment
  • a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that the vector or composition is not part of its natural environment.
  • isolated are: naturally occurring chromosomes
  • chromosome spreads e.g., chromosome spreads
  • artificial chromosome libraries e.g., genomic libraries
  • cDNA libraries that exist either as an in vitro nucleic acid preparation or as a transfected/transformed host cell preparation, wherein the host cells are either an in vitro heterogeneous preparation or plated as a heterogeneous population of single colonies.
  • a 5' EST makes up less than 5% (or alternatively 1%, 2%, 3%, 4%, 10%, 25%, 50%, 75%, or 90%, 95%, or 99%) of the number of nucleic acid inserts in the vector molecules.
  • whole cell genomic DNA or whole cell RNA preparations including said whole cell preparations which are mechanically sheared or enzymatically digested.
  • whole cell preparations as either an in vitro preparation or as a heterogeneous mixture separated by electrophoresis (including blot transfers of the same) wherein the polynucleotide of the invention have not been further separated from the heterologous polynucleotides in the electrophoresis medium (e.g., further separating by excising a single band from a heterogeneous band population in an agarose gel or nylon blot).
  • primer denotes a specific oligonucleotide sequence that is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence.
  • a primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.
  • probe denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., PNA as defined hereinbelow) which can be used to identify a specific polynucleotide sequence present in a sample, said nucleic acid segment comprising a nucleotide sequence complementary to the specific polynucleotide sequence to be identified.
  • polypeptide refers to a polymer of amino acids without regard to the length of the polymer.
  • peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not specify or exclude post-expression modifications of polypeptides.
  • polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, phosphate groups, lipid groups and the like are expressly encompassed by the term polypeptide.
  • polypeptides which contain one or more analogs of an amino acid (including, for example, non-naturally occurring amino acids, amino acids which only occur naturally in an unrelated biological system, modified amino acids from mammalian systems etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • GMG-1 refers genetically to the murine or human GMG-1, unless otherwise specified.
  • the compounds/polypeptides of the invention are capable of modulating the partitioning of dietary lipids between the liver and peripheral tissues, and are thus believed to treat "diseases involving the partitioning of dietary lipids between the liver and peripheral tissues.
  • peripheral tissues is meant to include muscle and adipose tissue.
  • the compounds/polypeptides of the invention partition the dietary lipids toward the muscle.
  • the dietary lipids are partitioned toward the adipose tissue, hi other preferred embodiments, the dietary lipids are partitioned toward the liver.
  • the compounds/polypeptides of the invention increase or decrease the oxidation of dietary lipids, preferably free fatty acids (FFA) by the muscle.
  • Dietary lipids include, but are not limited to triglycerides and free fatty acids.
  • Preferred diseases believed to involve the partitioning of dietary lipids include obesity and obesity-related diseases and disorders such as obesity, impaired glucose tolerance, insulin resistance, atherosclerosis, atheromatous disease, heart disease, hypertension, stroke, Syndrome X, Noninsulin Dependent Diabetes Mellitus (NIDDM, or Type II diabetes) and Insulin Dependent Diabetes Mellitus (IDDM or Type I diabetes).
  • Diabetes-related complications to be treated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy, and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia and hyperuricemia.
  • metabolic-related diseases or disorders of the invention including cachexia, wasting, AIDS-related weight loss, cancer-related weight loss, anorexia, and bulimia.
  • heterologous when used herein, is intended to designate any polypeptide or polynucleotide other than an GMG-1 or GMG-1 polypeptide or a polynucleotide encoding a gGMG- 1 polypeptide of the present invention.
  • the terms “comprising”, “consisting of and “consisting essentially of are defined according to their standard meaning. A defined meaning set forth in the M.P.E.P. controls over a defined meaning in the art and a defined meaning set forth in controlling Federal Circuit case law controls over a meaning set forth in the M.P.E.P. With this in mind, the terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term.
  • host cell recombinant for a particular polynucleotide of the present invention, means a host cell that has been altered by the hands of man to contain said polynucleotide in a way not naturally found in said cell.
  • said host cell may be transiently or stably transfected or transduced with said polynucleotide of the present invention.
  • BMI body mass index (morbid obesity) and is kg/m 2 .
  • Waist circumference can also be used to indicate a risk of metabolic complications where in men a circumference of greater than or equal to 94 cm indicates an increased risk, and greater than or equal to 102 cm indicates a substantially increased risk. Similarly for women, greater than or equal to 88 cm indicates an increased risk, and greater than or equal to 88 cm indicates a substantially increased risk.
  • the term "energy intake” as used herein is defined as the energy introduced into an individual from total caloric intake, i.e., the total energy from food and liquid diet.
  • the term “energy expenditure” as used herein is defined as total energy expenditure (TEE), which includes resting energy expenditure (REE), the thermic effect of feeding (TEF), and activities such as exercise. Both “energy intake” and “energy expenditure” are defined by Rosenbaum et al. [Am J Clin Nutr (2000) Jun;71(6):1421-32), which is hereby incorporated by reference in its entirety].
  • the term “maintenance of weight loss” as used herein is defined as sustaining a stable weight in an individual that is 10-20% below the initial, obese weight of the individual.
  • the new maintained weight after weight loss is a healthy weight (as defined herein).
  • the individual has a BMI of at least 20 and no more than 25.
  • the individual may have a BMI of at least 20.
  • a measured amount of glucose is given to the patient and blood glucose levels measured regular intervals, usually every half hour for the first two hours and every hour thereafter, hi a "normal" or non-IGT individual, glucose levels rise during the first two hours to a level less than 140 mg/dl and then drop rapidly.
  • the blood glucose levels are higher and the drop-off level is at a slower rate.
  • Insulin-Resistance Syndrome is intended to encompass the cluster of abnormalities resulting from an attempt to compensate for insulin resistance that sets in motion a series of events that play an important role in the development of both hypertension and coronary artery disease (CAD), such as premature atherosclerotic vascular disease.
  • CAD coronary artery disease
  • the invention provides methods for reducing and/or preventing the appearance of insulin-resistance syndrome.
  • PCOS polycystic ovary syndrome
  • insulin resistance is intended to encompass the usual diagnosis of insulin resistance made by any of a number of methods, such as the intravenous glucose tolerance test or measurement of the fasting insulin level. It is well known that there is an excellent correlation between the height of the fasting insulin level and the degree of insulin resistance. Therefore, one could use elevated fasting insulin levels as a surrogate marker for insulin resistance for the purpose of identifying which normal glucose tolerance (NGT) individuals have insulin resistance. Another way to do this is to follow the approach as disclosed in The New England
  • the target of the freatment according to the present invention can be defined as NGT individuals who are obese or who have fasting hyperinsulinemia, or who have both.
  • a diagnosis of insulin resistance can also be made using the euglycemic glucose clamp test.
  • This test involves the simultaneous administration of a constant insulin infusion and a variable rate glucose infusion. During the test, which lasts 3-4 hours, the plasma glucose concentration is kept constant at euglycemic levels by measuring the glucose level every 5-10 minutes and then adjusting the variable rate glucose infusion to keep the plasma glucose level unchanged. Under these circumstances, the rate of glucose entry into the bloodstream is equal to the overall rate of glucose disposal in the body. The difference between the rate of glucose disposal in the basal state (no insulin infusion) and the insulin infused state, represents insulin mediated glucose uptake.
  • NTDDM Noninsulin Dependent Diabetes Mellitus
  • response to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues refer to drug efficacy, including but not limited to, ability to metabolize a compound, ability to convert a pro-drug to an active drug, and the pharmacokmetics (abso ⁇ tion, distribution, elimination) and the pharmacodynamics (receptor-related) of a drug in an individual.
  • side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues refer to adverse effects of therapy resulting from extensions of the principal pharmacological action of the drug or to idiosyncratic adverse reactions resulting from an interaction of the drug with unique host factors.
  • Side effects to an agent acting on the partitioning of dietary lipids between the liver and peripheral tissues can include, but are not limited to, adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.
  • adverse reactions such as dermatologic, hematologic or hepatologic toxicities and further includes gastric and intestinal ulceration, disturbance in platelet function, renal injury, nephritis, vasomotor rhinitis with profuse watery secretions, angioneurotic edema, generalized urticaria, and bronchial asthma to laryngeal edema and bronchoconstriction, hypotension, and shock.
  • GMG-1 -related diseases and disorders refers to any disease or disorder comprising an aberrant functioning of GMG- 1 , or which could be freated or prevented by modulating GMG-1 levels or activity.
  • "Aberrant functioning of GMG-1” includes, but is not limited to, aberrant levels of expression of GMG-1 (either increased or decreased, but preferably decreased), aberrant activity of GMG-1 (either increased or decreased), and aberrant interactions with ligands or binding partners (either increased or decreased).
  • aberrant is meant a change from the type, or level of activity seen in normal cells, tissues, or patients, or seen previously in the cell, tissue, or patient prior to the onset of the illness.
  • these GMG-1-related diseases and disorders include obesity and the obesity-related diseases and disorders described previously.
  • cosmetic treatments is meant to include treatments with compounds or polypeptides of the invention that increase or decrease the body mass of an individual where the individual is not clinically obese or clinically thin.
  • these individuals have a body mass index (BMI) below the cut-off for clinical obesity (e.g. below 25 kg/m 2 ) and above the cut-off for clinical thinness (e.g. above 18.5 kg/m 2 ).
  • these individuals are preferably healthy (e.g. do not have an obesity-related disease or disorder of the invention).
  • Cosmetic treatments are also meant to encompass, in some circumstances, more localized increases in adipose tissue, for example, gains or losses specifically around the waist or hips, or around the hips and thighs, for example. These localized gains or losses of adipose tissue can be identified by increases or decreases in waist or hip size, for example.
  • in need of freatment refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, etc in the case of humans; veterinarian in the case of animals, including non-human mammals) that an individual or animal requires or will benefit from freatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that include the knowledge that the individual or animal is ill, or will be ill, as the result of a condition that is treatable by the compounds of the invention.
  • a caregiver e.g. physician, nurse, nurse practitioner, etc in the case of humans; veterinarian in the case of animals, including non-human mammals
  • perceives a need for treatment refers to a sub-clinical determination that an individual desires to reduce weight for cosmetic reasons as discussed under “cosmetic treatment” above.
  • the term “perceives a need for treatment” in other embodiments can refer to the decision that an owner of an animal makes for cosmetic freatment of the animal.
  • the term "individual” or “patient” as used herein refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans. The term may specify male or female or both, or exclude male or female.
  • non-human animal refers to any non-human vertebrate, including birds and more usually mammals, preferably primates, animals such as swine, goats, sheep, donkeys, horses, cats, dogs, rabbits or rodents, more preferably rats or mice. Both the terms “animal” and “mammal” expressly embrace human subjects unless preceded with the term "non-human”.
  • gGMG-1 a fragment of GMG-1, called gGMG-1, is able to significantly reduce the postprandial response of plasma free fatty acids, glucose, and triglycerides in mice fed a high fat/sucrose meal. There was no significant effect on leptin, insulin or glucagon levels.
  • gGMG-1 was found to increase muscle free fatty acid oxidation in vitro and ex vivo. Further, gGMG-1 was shown to decrease and then to prevent an increase in weight gain in mice that had been fed a high fat/sucrose diet for 19 days.
  • GMG-1 Polypeptide Fragments of the Invention GMG-1 polypeptide fragments that have measurable activity in vitro and in vivo have been identified. These activities include, but are not limited to, reduction of the postprandial response of plasma free fatty acids, glucose, and triglycerides in mice fed a high fat/sucrose meal (Example 8), increase in muscle free fatty acid oxidation in vitro and ex vivo (Example 12), and sustained weight loss in mice on a high fat/sucrose diet (Example 14).
  • Other assays for GMG-1 polypeptide fragment activity in vitro and in vivo are also provided (Examples 4, 7, 9, 11, 13, for example), and equivalent assays can be designed by those of ordinary skill in the art.
  • the "intact" or “full-length” GMG-1 polypeptide does not have either the in vivo or the in vitro activities that have been identified for gGMG-1 polypeptide fragments of the invention. In most cases, the activities are either not present or at a minimum are undetectable over confrol values in the assays used. In other cases, the activities can be measured, but are present either at extremely reduced levels and/or require significantly more protein on a molar basis compared with the gGMG-1 polypeptide fragments of the invention (see, e.g. Example 10).
  • gGMG-1 polypeptide fragments refers to polypeptide fragments comprised of the globular domain and is thus a narrower term than “GMG-1 polypeptide fragments".
  • fragment means a polypeptide having a sequence that is entirely the same as part, but not all, of an intact or full-length GMG-1 polypeptide. Such fragments may be "free-standing” (i.e. not part of or fused to other polypeptides), or one or more fragments may be present in a single polypeptide.
  • gGMG-1 fragments are contiguous fragments of the full-length GMG-1 polypeptide unless otherwise specified.
  • significantly greater refers to a comparison of the activity of an non-multimeric gGMG-1 polypeptide fragment homotrimer in an obesity-related assay compared with the activity of multimers of gGMG-1 polypeptide fragment homofrimer in the same assay.
  • significantly as used herein is meant statistically significant as it is typically determined by those with ordinary skill in the art. For example, data are typically calculated as a mean ⁇ SEM, and a p-value ⁇ 0.05 is considered statistically significant. Statistical analysis is typically done using either the unpaired Student's t test or the paired Student's t test, as appropriate in each study.
  • free fatty acid oxidation is measured in cells in vitro or ex vivo, preferably in muscle cells or tissue of non-human animals, preferably mice.
  • weight modulation is measured in human or non-human animals, preferably rodents (rats or mice), primates, canines, felines or procines on a high fat/sucrose diet.
  • "obesity-related activity” includes other activities not specifically identified herein.
  • preferred GMG-1 polypeptide fragments of the invention would have a significant change in at least one of the measurable parameters selected from the group consisting of an increase in LSR activity, an increase in leptin activity and an increase in lipoprotein activity.
  • LSR activity is meant expression of LSR on the surface of the cell, or in a particular conformation, as well as its ability to bind, uptake, and degrade leptin and lipoprotein.
  • leptin activity is meant its binding, uptake and degradation by LSR, as well as its transport across a blood brain barrier, and potentially these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.
  • lipoprotein activity is meant its binding, uptake and degradation by LSR, as well as these occurrences where LSR is not necessarily the mediating factor or the only mediating factor.
  • N-terminal putative signal sequence about from amino acids 1-15 of SEQ ID Nos:2 or 4;
  • collagen residues is used in the manner standard in the art to mean the amino acid triplet glycine, X, Y, where X and Y can be any amino acid.
  • the GMG-1 polypeptide fragments of the present invention are preferably provided in an isolated form, and may be partially or substantially purified.
  • a recombinantly produced version of an GMG-1 polypeptide fragment can be substantially purified by the one-step method described by Smith et al. ((1988) Gene 67(l):31-40) or by the methods described herein or known in the art (see, e.g., Examples 1-3).
  • Fragments of the invention also can be purified from natural or recombinant sources using antibodies directed against the polypeptide fragments of the invention by methods known in the art of protein purification.
  • GMG-1 polypeptide fragments of the invention involving a partial purification of or selection for the GMG-1 polypeptide fragments are also specifically contemplated. These crude preparations are envisioned to be the result of the concentration of cells expressing GMG-1 polypeptide fragments with perhaps a few additional purification steps, but prior to complete purification of the fragment.
  • the cells expressing GMG-1 polypeptide fragments are present in a pellet, they are lysed, or the crude polypeptide is lyophilized, for example.
  • gGMG-1 polypeptide fragments, and polynucleotides encoding the same can be any integer in length from 6 consecutive amino acids to 1 amino acid less than a full-length GMG-1 polypeptide.
  • a gGMG-1 polypeptide fragment can be any integer of consecutive amino acids from 6 to 243; for mouse GMG-1 of SEQ ID NO:4, a gGMG-1 polypeptide fragment can be any integer of consecutive amino acids from 6 to 246, for example.
  • integers include, but are not limited to: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 11
  • Each GMG-1 fragment as described above can be further specified in terms of its N-terminal and C-terminal positions. For example, every combination of a N-terminal and C-terminal position that fragments of from 6 contiguous amino acids to 1 amino acid less than the full-length GMG-1 polypeptide could occupy, on any given intact and contiguous full-length GMG-1 polypeptide sequence are included in the present invention. Thus, a 6 consecutive amino acid fragment could occupy positions selected from the group consisting of 1-6, 2-7, 3-8, 4-9, 5-10, 6-11, 7-12, 8-13, 9- 14, 10-15, 11-16, 12-17, 13-18, 14-19, 15-20, 16-21, 17-22, 18-23, 19-24, 20-25, 21-26, 22-27, 23-
  • positions occupied by fragments of 6 to next to the last amino acid consecutive amino acids in SEQ ID NOs: 2 or 4 are included in the present invention and can also be immediately envisaged based on these two examples and therefore are not individually listed solely for the pu ⁇ ose of not unnecessarily lengthening the specification.
  • positions occupied by fragments of 6 consecutive amino acids to 1 amino acid less than any other full length GMG-l_polypeptide can also be envisaged based on these two examples and therefore are not individually listed solely for the pu ⁇ ose of not unnecessarily lengthening the specification.
  • polypeptide fragments of SEQ ID NO:2 are selected from the group consisting of fragments comprising any 50 consecutive amino acids numbered from 1-50, 2-51, 3-52, 4-53, 5-54, 6-55, 7-56, 8-57, 9-58, 10-59, 11-60, 12-61, 13- 62, 14-63, 15-64, 16-65, 17-66, 18-67, 19-68, 20-69, 21-70, 22-71, 23-72, 24-73, 25-74, 26-75, 27- 76, 28-77, 29-78, 30-79, 31-80, 32-81, 33-82, 34-83, 35-84, 36-85, 37-86, 38-87, 39-88, 40-89, 41- 90, 42-91, 43-92, 44-93, 45-94, 46-95, 47-96, 48-97, 49-98, 50-99, 51-100, 52-101, 53-102, 54-103,
  • polypeptide fragments of SEQ ID NO: 2 are selected from the group consisting of fragments comprising any 100 consecutive amino acids numbered from 1-100, 2-101, 3-102, 4-103, 5-104, 6-105, 7-106, 8-107, 9-108, 10-109, 11- 110, 12-111, 13-112, 14-113, 15-114, 16-115, 17-116, 18-117, 19-118, 20-119, 21-120, 22-121, 23- 122, 24-123, 25-124, 26-125, 27-126, 28-127, 29-128, 30-129, 31-130, 32-131, 33-132, 34-133, 35- 134, 36-135, 37-136, 38-137, 39-138, 40-139, 41-140, 42-141, 43-142, 44-143, 45-144, 46-145, 47- 146, 48-147, 49-148, 50-149, 51-150, 52-151, 53-152, 54-153, 55
  • a 238 consecutive amino acid fragment could occupy positions selected from the group consisting of 1-238, 2-239, 3-240, 4-241, 5-242, 6-243 and 7-244 of SEQ TD NO:2. Similarly, the positions occupied by all the other fragments of sizes between 6 amino acids and 284 amino acids on
  • SEQ ID NO:2 are included in the present invention and can also be immediately envisaged based on the examples for fragments of 6, 50, 100 or 284 consecutive amino acids listed above, and therefore, are not individually listed solely for the pu ⁇ ose of not unnecessarily lengthening the specification.
  • positions occupied by fragments of 6 to 217 consecutive amino acids on SEQ ID NO:4 are included in the present invention and can also be immediately envisaged based on these two examples and therefore are not individually listed solely for the pu ⁇ ose of not unnecessarily lengthening the specification.
  • gGMG-1 polypeptide fragments, and polynucleotides encoding the same, having unexpected activity are selected from amino acids numbered from 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285,
  • gGMG-1 polypeptide fragments, and polynucleotides encoding the same, having unexpected activity are selected from amino acids numbered from 16-217, 17-217, 18-217, 19-217, 20-217, 21-217, 22- 217, 23-217, 24-217, 25-217, 26-217, 27-217, 28-217, 29-217, 30-217, 31-217, 32-217, 33-217, 34- 217, 35-217, 36-217, 37-217, 38-217, 39-217, 40-217, 41-217, 42-217, 43-217, 44-217, 16-217, 17- 217, 18-217, 19-217, 20-217, 21-217, 22-217, 23-217, 24-217, 25-217, 26-217, 27-217, 28-217, 29- 217, 30-217, 31-217, 32-217, 33-217, 34-217, 35-217, 36-217, 37-217, 38-217, 39-217, 40-2
  • gGMG-1 polypeptide fragments, and polynucleotides encoding the same, having unexpected activity are selected from amino acids numbered from 45-285, 47-285, 126-285, 127-285, 132-285, 133-285, or 134-285 of SEQ ID NO:2.
  • gGMG-1 polypeptide fragments, and polynucleotides encoding the same, having unexpected activity are selected from amino acids numbered from 45-217, 47-217, or 88-217 of SEQ ID NO:4.
  • the gGMG-1 polypeptide fragments of the present invention may alternatively be described by the formula "n to c" (inclusive); where "n” equals the N-terminal most amino acid position (as defined by the sequence listing) and “c” equals the C-terminal most amino acid position (as defined by the sequence listing) of the polypeptide; and further where "n” equals an integer between 1 and the number of amino acids of the full length polypeptide sequence of the present invention minus 5 (280 for SEQ ID NO:2 and 212 for SEQ ID NO:4); and where "c” equals an integer between 6 and the number of amino acids of the full-length polypeptide sequence (285 for SEQ ID NO:2 and 217 for SEQ TD NO:4); and where "n” is an integer smaller then "c" by at least 6.
  • n is any integer selected from the list consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111
  • n and c positions are included as specific embodiments of the invention.
  • the formula "n” to “c” may be modified as '"nl - n2" to "cl - c2'", wherein “nl - n2" and “cl - c2" represent positional ranges selected from any two integers above which represent amino acid positions of the sequence listing.
  • Alternative formulas include '"nl -n2" to "c"' and
  • the present invention also provides for the exclusion of any individual fragment specified by N-terminal and C-terminal positions or of any fragment specified by size in amino acid residues as described above.
  • any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may be excluded as individual species.
  • any number of fragments specified by N-terminal and C-terminal positions or by size in amino acid residues as described above may make up a polypeptide fragment in any combination and may optionally include non-GMG-1 polypeptide sequence as well.
  • gGMG-1 polypeptide fragments of the invention include variants, fragments, analogs and derivatives of the gGMG-1 polypeptide fragments described above, including modified gGMG-1 polypeptide fragments.
  • Proteolytic cleavage of full length GMG-1 polypeptides of the invention in vivo is believed to be subject to complex regulation that facilitates the appropriate and effective generation of GMG- 1 polypeptide fragments of the invention comprised of all or part of the globular C-terminal Clq homology region and having unexpected lipid partitioning, lipid metabolism, and insulin-like activity.
  • Said proteolytic cleavage is regulated in part by selective presentation of protease cleavage sites through alternative splicing.
  • Said proteolytic cleavage is further regulated at the level of the protease, for example at the level of tissue distribution of the protease and at the level of amount of the protease, which itself can be regulated by physiological signals such as those associated with inflammation.
  • Particularly preferred GMG-1 polypeptide fragments of the invention comprised of all or part of the globular C-terminal Clq homology region and having unexpected lipid partitioning, lipid metabolism, and insulin-like activity are said GMG-1 polypeptide fragments of SEQ ID NOs: 2, or 4 believed to be generated proteolytically in vivo.
  • Particularly preferred is GMG-1 fragment of about amino acids 47-217 of SEQ
  • GMG-1 fragment of about amino acids 47-285 of SEQ ID NO: 2 made by matrix metalloproteinase-1 (MMP-1) cleavage of SEQ ID NO: 2 at about position 47.
  • GMG-1 polypeptides of the invention include variants, fragments, analogs and derivatives of the GMG-1 polypeptides described above, including modified GMG-1 polypeptides.
  • the invention further includes variants of gGMG-1 polypeptide fragments that have obesity-related activity as described above.
  • Such variants include GMG-1 fragment sequences with one or more amino acid deletions, insertions, inversions, repeats, and substitutions either from natural mutations or human manipulation selected according to general rules known in the art so as to have little effect on activity. Guidance concerning how to make phenotypically silent amino acid substitutions is provided below.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, Val, Leu and Phe; interchange of the hydroxyl residues Ser and Thr; exchange of the acidic residues Asp and Glu; substitution between the amide residues Asn and Gin; exchange of the basic residues Lys and Arg; and replacements among the aromatic residues Phe, Tyr.
  • amino acids in the gGMG-1 polypeptide fragment sequences of the invention that are essential for function can also be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham, et al. (1989) Science 244(4908): 1081-5). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for obesity-related activity using assays as described above. Of special interest are substitutions of charged amino acids with other charged or neutral amino acids that may produce proteins with highly desirable improved characteristics, such as less aggregation.
  • Aggregation may not only reduce activity but also be problematic when preparing pharmaceutical or physiologically acceptable formulations, because aggregates can be immunogenic (see, e.g., Pinckard, et al., (1967) Clin. Exp. Immunol 2:331-340; Robbins, et al., (1987) Diabetes Jul;36(7):838-41; and Cleland, et al., (1993) Crit Rev Ther Drug Carrier Syst. 10(4):307-77).
  • the fragment, derivative, analog, or homolog of the gGMG-1 fragment of the present invention may be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code (i.e.
  • the gGMG-1 fragment may be a non-naturally occurring amino acid); or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the gGMG-1 fragment is fused with another compound, such as a compound to increase the half-life of the fragment (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the above form of the fragment , such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the fragment or a pro-protein sequence.
  • additional amino acids are fused to the above form of the fragment , such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the fragment or a pro-protein sequence.
  • a further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of a gGMG-1 polypeptide fragment having an amino acid sequence which contains at least one conservative amino acid substitution, but not more than 50 conservative amino acid substitutions, not more than 40 conservative amino acid substitutions, not more than 30 conservative amino acid substitutions, and not more than 20 conservative amino acid substitutions. Also provided are polypeptides which comprise the amino acid sequence of a gGMG-1 fragment, having at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative amino acid substitutions.
  • a further embodiment of the invention relates to a GMG-1 polypeptide fragment made resistant to dipeptidyl peptidase cleavage through N-terminal modification of said polypeptide fragment.
  • said GMG-1 polypeptide fragment is selected from amino acids 47-285, 127-285 or 134-285 of SEQ ID NO: 2, or amino acids 47-217 of SEQ ID NO: 4.
  • said dipeptidyl peptidase cleavage leads to removal of the N-terminal dipeptide PP, GP or EP by dipeptidyl peptidase from said preferred fragment, hi another preferred embodiment, said dipeptidyl peptidase is human plasma comprised of dipeptidyl peptidase.
  • said dipeptidyl peptidase is selected from but not restricted to human CD26 and human Attractin. In a further preferred embodiment, said dipeptidyl peptidase is selected from soluble human CD26 or soluble human Attractin.
  • said N-terminal modification is selected from but not restricted to glycation [Harte (2001) Regulatory Peptides 96:95-104 which disclosure is hereby inco ⁇ orated by reference in its entirety], N-methylation, alpha-methylation, desamidation [Gallwitz (2000) Regulatory Peptides 86:103-111 which disclosure is hereby inco ⁇ orated by reference in its entirety], or alternation of the chirality of one or more N- terminal amino acids [Siegel (1999) European Journal of Clinical Investigation 29:610-614 which disclosure is hereby inco ⁇ orated by reference in its entirety].
  • the invention also encompasses a GMG-1 polypeptide fragment or a variant thereof that has been made resistant to dipeptidyl peptidase cleavage through N-terminal modification of said polypeptide fragment.
  • a modified gGMG-1 fragment of the invention is a polypeptide that is resistant to proteolysis, for example a gGMG-1 fragment in which a -CONH- peptide bond is modified and replaced by one or more of the following: a (CH2NH) reduced bond; a (NHCO) retro inverso bond; a (CH2-0) methylene-oxy bond; a (CH2-S) thiomethylene bond; a
  • the invention also encompasses a gGMG-1 fragment or a variant thereof in which at least one peptide bond has been modified as described above.
  • amino acids have chirality within the body of either L or D. i some embodiments it is preferable to alter the chirality of the amino acids in the gGMG-1 polypeptide fragments of the invention in order to extend half-life within the body.
  • one or more of the amino acids are preferably in the L configuration. In other embodiments, one or more of the amino acids are preferably in the D configuration.
  • polypeptides of the present invention also include polypeptides having an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a gGMG-1 fragment as described above.
  • a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a gGMG-1 fragment amino acid sequence is meant that the amino acid sequence is identical to the gGMG-1 polypeptide fragment sequence except that it may include up to five amino acid alterations per each 100 amino acids of the gGMG-1 polypeptide fragment amino acid sequence.
  • the reference sequence is the gGMG-1 polypeptide fragment with a sequence corresponding to the sequence of the sequence listing.
  • polypeptide having an amino acid sequence at least 95% identical to a gGMG-1 fragment amino acid sequence up to 5% (5 of 100) of the amino acid residues in the sequence may be inserted, deleted, or substituted with another amino acid compared with the gGMG-1 polypeptide fragment sequence.
  • alterations may occur at the amino or carboxy termini or anywhere between those terminal positions, interspersed either individually among residues in the sequence or in one or more contiguous groups within the sequence.
  • any particular polypeptide is a percentage identical to a gGMG-1 fragment can be determined conventionally using known computer programs.
  • Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FAST A, TFASTA, and CLUSTALW (Pearson and Lipman, (1988) Proc Natl Acad Sci USA 85(8):2444-8; Altschul et al., (1990) J Mol Biol 215(3):403-410; Thompson et al., (1994) Nucleic Acids Res 22(2):4673-4680; Higgins et al, (1996) Meth Enzymol 266:383-402; Altschul et al, (1997) Nuc Acids Res 25:3389-3402; Altschul et al., (1993) Nature Genetics 3:266-272).
  • BLAST Basic Local Alignment Search Tool
  • BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database
  • BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database
  • TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands)
  • TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.
  • the BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as "high-scoring segment pairs," between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database.
  • High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art.
  • the scoring matrix used is the BLOSUM62 matrix (see, Gonnet et al., (1992) Science 256(5062): 1443-5; Henikoff and Henikoff (1993) Proteins 17(1):49-61).
  • the PAM or PAM250 matrices may also be used (See, e.g., Schwartz and Dayhoff, eds, (1978) Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation).
  • the BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user- specified percent homology.
  • the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (See, e.g., Karlin and Altschul, (1990) Proc Natl Acad Sci USA 87(6):2264-8).
  • the BLAST programs may be used with the default parameters or with modified parameters provided by the user. Preferably, the parameters are default parameters.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of
  • the results, in percent identity must be manually corrected because the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C- terminal of the subject sequence, that are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment.
  • This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This final percent identity score is what is used for the pu ⁇ oses of the present invention. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the pu ⁇ oses of manually adjusting the percent identity score. That is, only query amino acid residues outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100-residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not match/align with the first residues at the
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90-residue subject sequence is compared with a 100-residue query sequence. This time the deletions are internal so there are no residues at the N- or C-termini of the subject sequence, which are not matched/aligned with the query. In this case, the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected. No other manual corrections are made for the pu ⁇ oses of the present invention. Production
  • GMG-1 polypeptide fragments are discussed, gGMG-1 fragments are specifically intended to be included as a preferred subset of GMG-1 polypeptide fragments.
  • GMG-1 polypeptide fragments are preferably isolated from human or mammalian tissue samples or expressed from human or mammalian genes in human or mammalian cells.
  • the GMG-1 polypeptide fragments of the invention can be made using routine expression methods known in the art.
  • the polynucleotide encoding the desired polypeptide fragments is ligated into an expression vector suitable for any convenient host. Both eukaryotic and prokaryotic host systems are used in forming recombinant polypeptide fragments.
  • the polypeptide fragment is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use. Purification is by any technique known in the art, for example, differential extraction, salt fractionation, chromatography, centrifugation, and the like.
  • polypeptides of the invention are isolated from milk.
  • the polypeptides can be purified as full-length GMG-1 polypeptides, which can then be cleaved, if appropriate, in vitro to generate an GMG-1 fragment, or, alternatively, GMG-1 fragments themselves can be purified from the milk. Any of a large number of methods can be used to purify the present polypeptides from milk, including those taught in Protein Purification Applications, A Practical Approach (New Edition), Edited by Simon Roe, AEA Technology Products and Systems,
  • milk is centrifuged, e.g. at a relatively low speed, to separate the lipid fraction, and the aqueous supernatant is then centrifuged at a higher speed to separate the casein in the milk from the remaining, "whey" fraction.
  • whey aqueous supernatant
  • biomedical proteins are found in this whey fraction, and can be isolated from this fraction using standard chromatographic or other procedures commonly used for protein purification, e.g. as described elsewhere in the present application.
  • GMG-1 polypeptides are purified using antibodies specific to GMG-1 polypeptides, e.g. using affinity chromatography.
  • methods can be used to isolate particular GMG-1 fragments, e.g. electrophoretic or other methods for isolating proteins of a particular size.
  • the GMG-1 polypeptides isolating using these methods can be naturally occurring, as GMG-1 polypeptides have been discovered to be naturally present in the milk of mammals (see, e.g. Example 17), or can be the result of the recombinant production of the protein in the mammary glands of a non-human mammal, as described infra.
  • the GMG-1 fragment is produced as a fusion protein with a heterologous, antigenic polypeptide sequence, which antigenic sequence can be used to purify the protein, e.g., using standard immuno-affinity methodology.
  • proteins of the invention are extracted from cells or tissues of humans or non- human animals. Methods for purifying proteins are known in the art, and include the use of detergents or chaotropic agents to disrupt particles followed by differential extraction and separation of the polypeptides by ion exchange chromatography, affinity chromatography, sedimentation according to density, and gel electrophoresis.
  • Any GMG-1 fragment cDNA can be used to express GMG-1 polypeptide fragments.
  • the nucleic acid encoding the GMG-1 fragment to be expressed is operably linked to a promoter in an expression vector using conventional cloning technology.
  • the GMG-1 fragment cDNA insert in the expression vector may comprise the coding sequence for: the full-length GMG-1 polypeptide (to be later modified); from 6 amino acids to 1 amino acid less than the full-length GMG-1 polypeptide; a gGMG-1 fragment; or variants and % similar polypeptides.
  • the expression vector is any of the mammalian, yeast, insect or bacterial expression systems known in the art, some of which are described herein, and examples of which are given in the Examples (Examples 1-3).
  • Commercially available vectors and expression systems are available from a variety of suppliers including Genetics Institute (Cambridge, MA), Stratagene (La Jolla, California), Promega (Madison, Wisconsin), and Invitrogen (San Diego, California).
  • the codon context and codon pairing of the sequence can be optimized for the particular expression organism into which the expression vector is introduced, as explained by Hatfield, et al., US Patent Number 5,082,767, the disclosures of which are inco ⁇ orated by reference herein in their entirety.
  • nucleic acid encoding GMG-1 polypeptide fragments lacks a methionine to serve as the initiation site, an initiating methionine can be introduced next to the first codon of the nucleic acid using conventional techniques.
  • this sequence can be added to the construct by, for example, splicing out the Poly A signal from pSG5 (Stratagene) using Bgll and Sail restriction endonuclease enzymes and inco ⁇ orating it into the mammalian expression vector pXTl (Stratagene).
  • pXTl contains the LTRs and a portion of the gag gene from Moloney Murine Leukemia Virus. The position of the LTRs in the construct allow efficient stable transfection.
  • the vector includes the He ⁇ es Simplex Thymidine Kinase promoter and the selectable neomycin gene.
  • the nucleic acid encoding an GMG-1 fragment can be obtained by PCR from a vector containing the GMG-1 nucleotide sequence using oligonucleotide primers complementary to the desired GMG-1 cDNA and containing restriction endonuclease sequences for Pst I inco ⁇ orated into the 5' primer and BglTI at the 5' end of the corresponding cDNA 3' primer, taking care to ensure that the sequence encoding the GMG-1 fragment is positioned properly with respect to the poly A signal.
  • the purified fragment obtained from the resulting PCR reaction is digested with Pstl, blunt ended with an exonuclease, digested with Bgl II, purified and ligated to pXTl, now containing a poly A signal and digested with Bgi ⁇ .
  • Alternative methods are presented in Examples 1-3. Transfection of an GMG-1 fragment-expressing vector into mouse NTH 3T3 cells is one embodiment of introducing polynucleotides into host cells.
  • Introduction of a polynucleotide encoding a polypeptide into a host cell can be effected by calcium phosphate fransfection, DEAE-dexfran mediated transfection, cationic lipid-mediated fransfection, electroporation, fransduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al. ((1986) Methods in Molecular Biology, Elsevier Science Publishing Co., Inc., Amsterdam). It is specifically contemplated that the polypeptides of the present invention may in fact be expressed by a host cell lacking a recombinant vector. Methods of expressing GMG- 1 fragment of the invention in cells are described in Examples 1-3.
  • a polypeptide of this invention (i.e. a gGMG-1 fragment) can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the present invention can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
  • polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • GMG-1 globular domain comprising the polypeptides of the invention is non-glycosylated.
  • polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells.
  • N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
  • the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with the polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous polynucleotides.
  • endogenous genetic material e.g., coding sequence
  • genetic material e.g., heterologous polynucleotide sequences
  • heterologous confrol regions e.g., promoter and/or enhancer
  • endogenous polynucleotide sequences via homologous recombination
  • techniques known in the art may be used to operably associate heterologous confrol regions (e.g., promoter and/or enhancer) and endogenous polynucleotide sequences via homologous recombination, see, e.g., US Patent Number 5,641,670, issued June 24, 1997; International Publication No. WO 96/29411, published September 26, 1996; International Publication No. WO 94/12650, published August 4, 1994; Roller et al., (1989) Proc Natl Acad Sci USA 86(22):8932-5; Roller et al., (1989) Proc Natl Acad Sci USA 86(22):8927-31; and Zijlstra et al. (1989) Nature 342(6248):435-8; the disclosures of each of which are inco ⁇ orated
  • polypeptides of the invention can be chemically synthesized using techniques known in the art (See, e.g., Creighton, 1983 Proteins. New York, New York: W.H. Freeman and Company; and Hunkapiller et al., (1984) Nature 310(5973):105-11).
  • a relative short fragment of the invention can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the fragment sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D
  • the invention encompasses polypeptide fragments which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular Iigand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.
  • Additional post-translational modifications encompassed by the invention include, for example, N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
  • the polypeptide fragments may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the polypeptide.
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • polyethylene glycol molecules should be attached to the polypeptide with consideration of effects on functional or antigenic domains of the polypeptide.
  • attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein inco ⁇ orated by reference (coupling PEG to G-CSF), see also Malik et al. (1992) Exp Hematol 20(8): 1028-35, reporting pegylation of GM-CSF using fresyl chloride).
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules.
  • Preferred for therapeutic pu ⁇ oses is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N-terminus may be accomplished by reductive alkylation, which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • the invention features a method of reducing body mass comprising providing or administering to individuals in need of reducing body mass said pharmaceutical or physiologically acceptable composition described in the fifth aspect in combination with provision or administration of an antagonist of dipeptidyl peptidase cleavage of GMG-1 polypeptide fragment of the first aspect.
  • Preferred said antagonist is a peptidyl derivative of a diester of alpha- aminoalkylphosphonic acid (US Patent Number 5,543,396 which disclosure is hereby inco ⁇ orated by reference in its entirety). More preferred said peptidyl derivative is selected from Ala-Pro p (OZ) 2 , AcOH.Ala-Pip p (Oph) 2 , HCl.Ala-Pro p (Oph-4Cl) 2 , HCl.Ala-Pip p (Oph-4Cl) 2 , or 2HCl.Lys-Pip p (Oph- 4C1) 2 , where Z represents an aryl group, a substituted aryl group or a highly flourinated alkyl group, Pro p represents a proline phosphonate derivative, and Pip p represents piperidyl phosphonate (US Patent Number 5,543,396 which disclosure is hereby inco ⁇ orated by reference in its entirety).
  • Xaa is an amino acid
  • Z is a protecting group
  • Y' is one of various types of ring structures
  • Z may or may not be present and represents a protecting group, such as benzyloxycarbonyl;
  • Xaa represents alanine, methionine, arginine, phenylalanine, aspartic acid, proline, asparagine, serine, cysteine, threonine, glycine, tyrosine, glutamic acid, tryptophan, glutamine, valine, isoleucine, lysine, leucine, L-thioproline, L-homoproline, L-
  • Tic 1, 2,3 ,4,tetrahydroisoquinoline-3 -carboxylic acid
  • Tic L-2,3-dihydroindol-2-carboxylic acid
  • L- naphthylglycine L-phenylglycine
  • L-4-phenylproline O-benzyl tyrosine
  • omega-Z lysine or omega- acetyl lysine
  • Y' represents a pyrrolidide, a phosphonate or phosphinate derivative, or reduced peptide; or pharmaceutically acceptable salts thereof
  • N-(substituted glycyl)-2-cyanopyrrolidine (US Patent Number 6,166,063). More preferred said N-(substituted glycyl)-2-cyanopyrrolidine is selected from pyrrolidine, l-[[(3,5-dimethyl-l-adamantyl)amino]-acetyl]-2-cyano-, (S)-; pyrrolidine, l-[[(3-ethyl- l-adamantyl)amino]-acetyl]-2-cyano-, (S)-; pyrrolidine, l-[[(3-methoxy-l-adamantyl)amino]- acetyTJ-2-cyano-, (S)-; pyrrolidine, l-[[[3-[[[(t-butylamino)carbonyl]oxy]-l-adamantyl]amino]- acety
  • tetrahydroisoquinoline 3-carboxamide derivative of formula ##STR1## (US Patent Number 6,172,081). More preferred is said derivative and pharmaceutically acceptable salts thereof wherein X is CH 2 , S, O, or C(CH 3 ) 2 ; Ri and R 2 are independently hydrogen, hydroxy, alkyl, alkoxy, aralkoxy, or halogen (US Patent Number 6,172,081).
  • valine-pyrrolidide (Deacon (2001) Diabetes 50:1588- 1597 which disclosure is hereby inco ⁇ orated by reference in its entirety].
  • the polypeptide fragments of the invention may be in monomers or multimers. Most preferably, the polypeptide fragments of the invention are in homotrimers. Accordingly, the present invention relates to monomers and multimers of the polypeptide fragments of the invention, their preparation, and compositions (preferably, pharmaceutical or physiologically acceptable compositions) containing them. In specific embodiments, the polypeptides of the invention are homotrimers. In additional embodiments, the multimers of the invention comprise, consist essentially of, or consist of homofrimers. Multimers encompassed by the invention may be homomers or heteromers.
  • homomer refers to a multimer containing only polypeptides corresponding to the GMG-1 polypeptide fragments of the invention (including polypeptide fragments, variants, splice variants, and fusion proteins corresponding to these polypeptide fragments as described herein). These homomers may contain polypeptide fragments having identical or different amino acid sequences.
  • a homomer of the invention is a multimer containing only polypeptide fragments having an identical amino acid sequence. In another specific embodiment, a homomer of the invention is a multimer containing polypeptide fragments having different amino acid sequences.
  • the multimer of the invention is a homodimer (e.g., containing polypeptide fragments having identical or different amino acid sequences) or a homotrimer (e.g., containing polypeptide fragments having identical and/or different amino acid sequences).
  • the homomeric multimer of the invention is at least a homodimer, at least a homofrimer, or at least a homoteframer. More preferably, the homomeric multimer of the invention is a homofrimer. Further more preferably, said homofrimer of the invention is gGMG-1 polypeptide ' fragment homotrimer.
  • said gGMG-1 polypeptide fragment homofrimer of the invention has activity selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity. Also most preferably, said gGMG-1 polypeptide fragment homotrimer of the invention has activity selected from the group consisting of prevention of weight gain, weight reduction, and maintenance of weight loss.
  • the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., corresponding to different proteins or polypeptide fragments thereof) in addition to the polypeptides of the invention.
  • the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.
  • the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
  • Multimers of the invention maybe the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
  • homotrimers of the invention are formed when polypeptides of the invention contact one another in solution.
  • heteromultimers of the invention such as, for example, heterofrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.
  • multimers of the invention are formed by covalent associations with and/or between the polypeptides of the invention.
  • covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in the sequence listing, or contained in the polypeptide encoded by a deposited clone).
  • the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences, which interact in the native (i.e., naturally occurring) polypeptide.
  • the covalent associations are the consequence of chemical or recombinant manipulation.
  • covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a fusion protein of the invention.
  • said multimers of the invention are gGMG-1 polypeptide fragment homofrimers.
  • said gGMG-1 polypeptide fragment homofrimer of the invention has activity selected from the group consisting of lipid partitioning, lipid metabolism, and insulin-like activity. Also most preferably, said gGMG-1 polypeptide fragment homotrimer of the invention has activity selected from the group consisting of prevention of weight gain, weight reduction, and maintenance of weight loss.
  • covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925).
  • the covalent associations are between the heterologous sequence contained in an Fc fusion protein of the invention (as described herein).
  • covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein inco ⁇ orated by reference in its entirety).
  • Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.
  • Leucine zippers were originally identified in several DNA-binding proteins, and have since been found in a variety of different proteins (Landschulz et al., (1988) Genes Dev. Jul;2(7):786-800).
  • the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
  • leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby inco ⁇ orated by reference.
  • Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
  • Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.
  • Preferred leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.
  • One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. FEBS Letters (1994) 344(2-3): 191-5 and in U.S. patent application Ser. No.
  • the multimers of the invention may be generated using chemical techniques known in the art.
  • polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
  • multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
  • multimers of the invention may be generated using genetic engineering techniques known in the art.
  • polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
  • polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
  • Preferred polynucleotides are those that encode full-length GMG-1 and gGMG-1 polypeptide fragments of the invention.
  • the recombinant polynucleotides encoding full-length GMG-1 and gGMG-1 polypeptide fragments can be used in a variety of ways, including, but not limited to, expressing the polypeptide in recombinant cells for use in screening assays for antagonists and agonists of its activity as well as to facilitate its purification for use in a variety of ways including, but not limited to screening assays for agonists and antagonists of its activity, diagnostic screens, and raising antibodies, as well as freatment and/or prevention of obesity-related diseases and disorders and/or to reduce body mass.
  • polynucleotide of which they form a part or region.
  • gGMG-1 polynucleotide fragments may be comprised within a single polynucleotide.
  • the GMG-1 polynucleotides of the invention comprise from 18 consecutive bases to 18 consecutive bases less than the full-length polynucleotide sequence encoding the intact GMG-1 polypeptide, for example the full-length GMG-1 polypeptide polynucleotide sequences in SEQ ID NO: 1 or SEQ ID NO: 3.
  • the polynucleotide comprises at least 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480
  • nucleic acids comprise at least 18 nucleotides, wherein "at least 18" is defined as any integer between 18 and the integer representing the 3' most nucleotide position of the intact GMG-1 polypeptide cDNA as set forth in the sequence listing (SEQ ID NO:l or SEQ ID NO: 3) or elsewhere herein.
  • polynucleotide fragments of the present invention may alternatively be described by the formula "x to y"; where "x" equals the 5' most nucleotide position and “y” equals the 3' most nucleotide position of the polynucleotide; and further where "x” equals an integer between 1 and the number of nucleotides of the polynucleotide sequence of the present invention minus 18, and where "y” equals an integer between 19 and the number of nucleotides of the polynucleotide sequence of the present invention; and where "x" is an integer smaller then "y” by at least 18.
  • the present invention also provides for the exclusion of any species of polynucleotide fragments of the present invention specified by 5' and 3' positions or polynucleotides specified by size in nucleotides as described above. Any number of fragments specified by 5' and 3' positions or by size in nucleotides, as described above, may be excluded.
  • the gGMG-1 polynucleotide fragments of the invention comprise from 18 consecutive bases to the full-length polynucleotide sequence encoding the gGMG-1 fragments described in Section ⁇ of the Preferred Embodiments of the Invention.
  • futher preferred nucleic acids comprise at least 18 nucleotides, wherein "at least 18" is defined as any integer between 18 and the integer corresponding to the 3 ' most nucleotide position of a gGMG-1 fragment cDNA herein.
  • nucleic acid fragments at least 18 nucleotides in length, as described above, that are further specified in terms of their 5' and 3' position.
  • the 5' and 3' positions are represented by the position numbers set forth in the sequence listing below.
  • position 1 is defined as the
  • polynucleotide fragments of the present invention may alternatively be described by the formula "x to y"; where "x" equals the 5' most nucleotide position and “y” equals the 3' most nucleotide position of the polynucleotide; and further where "x” equals an integer between 1 and the number of nucleotides of the gGMG-1 polynucleotide sequence of the present invention minus 18, and where "y” equals an integer between 9 and the number of nucleotides of the gGMG-1 polynucleotide sequence of the present invention; and where "x” is an integer smaller than "y” by at least 18. .
  • the present invention also provides for the exclusion of any species of polynucleotide fragments of the present invention specified by 5' and 3' positions or polynucleotides specified by size in nucleotides as described above. Any number of fragments specified by 5' and 3' positions or by size in nucleotides, as described above, may be excluded.
  • Variants In other preferred embodiments, variants of gGMG-1 polynucleotides encoding gGMG-1 polypeptide fragments are envisioned.
  • Variants of polynucleotides, as the term is used herein are polynucleotides whose sequence differs from a reference polynucleotide.
  • a variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.
  • Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.
  • Polynucleotide variants that comprise a sequence substantially different from those described above but that, due to the degeneracy of the genetic code, still encode gGMG-1 polypeptide fragments of the present invention are also specifically envisioned. It would also be routine for one skilled in the art to generate the degenerate variants described above, for instance, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by other mammalian or bacterial host cells).
  • variant polynucleotides may occur naturally, such as a natural allelic variant, or by recombinant methods.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism (See, e.g., B. Lewin, (1990) Genes TV, Oxford University Press, New York).
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • Such nucleic acid variants include those produced by nucleotide substitutions, deletions, or additions. The substitutions, deletions, or additions may involve one or more nucleotides. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of a gGMG-1 polypeptide fragment of the invention. Also preferred in this regard are conservative substitutions.
  • Nucleotide changes present in a variant polynucleotide are preferably silent, which means that they do not alter the amino acids encoded by the polynucleotide. However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence.
  • preferred gGMG-1 polypeptide fragments include those that retain one or more obesity-related activity as described in Section I of the Preferred Embodiments of the Invention.
  • “retain the same activities” is meant that the activity measured using the polypeptide encoded by the variant gGMG-1 polynucleotide in assays is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%, and not more than 101%, 102%, 103%, 104%, 105%, 110%, 115%, 120%) or 125% of the activity measured using a gGMG-1 fragment described in the Examples Section herein.
  • the activity being “decreased” is meant that the activity measured using the polypeptide encoded by the variant gGMG-1 polynucleotide in assays is decreased by at least 25%>, 30%, 35%, 40%, 45%, or 50% of the activity measured using a gGMG-1 fragment described in the Examples Section herein.
  • the present invention is further directed to nucleic acid molecules having sequences at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences of SEQ ID NO: 1 or SEQ ID NO: 3 or fragments thereof that encode a polypeptide having obesity- related activity as described in Section I of the Preferred Embodiments of the Invention.
  • nucleic acid molecules at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequences shown in SEQ ID NO: lor SEQ ID NO:3 or fragments thereof will encode a polypeptide having biological activity.
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
  • nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having biological activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly affect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described previously in Section I of the Preferred Embodiments of the Invention.
  • the methods of determining and defining whether any particular nucleic acid molecule or polypeptide is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the present invention can be done by using known computer programs.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., ((1990) Comput Appl Biosci 6(3):237-45). In a sequence alignment the query and subject sequences are both DNA sequences.
  • RNA sequence can be compared by first converting U's to T's. The result of said global sequence alignment is in percent identity.
  • This percentage is then subfracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This corrected score is what is used for the pu ⁇ oses of the present invention. Only nucleotides outside the 5' and 3' nucleotides of the subject sequence, as displayed by the FASTDB alignment, which are not matched aligned with the query sequence, are calculated for the pu ⁇ oses of manually adjusting the percent identity score.
  • the "HA” tag is another peptide useful for purification which co ⁇ esponds to an epitope derived from the influenza hemagglutinin protein (See, Wilson et al., (1984) Cell 37(3):767-78).
  • other such fusion proteins include gGMG-1 fragment cDNA fused to Fc at the N- or C-terminus.
  • the present invention relates to recombinant vectors comprising any one of the polynucleotides described herein.
  • a recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid, or even a linear DNA molecule which may consist of a chromosomal, non- chromosomal, semi-synthetic or synthetic DNA.
  • a recombinant vector can comprise a transcriptional unit comprising an assembly of :
  • recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence.
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of the translated protein into the periplasmic space or the extracellular medium.
  • preferred vectors will comprise an origin of replication in the desired host, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5 '-flanking non-transcribed sequences.
  • DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
  • the suitable promoter regions used in the expression vectors of the present invention are chosen taking into account the cell host in which the heterologous gene is expressed.
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell.
  • a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the confrol of a promoter that is capable of being expressed in a human cell, such as, for example, a human or a viral promoter.
  • the promoter used may be constitutive or inducible.
  • a suitable promoter may be heterologous with respect to the nucleic acid for which it controls the expression or alternatively can be endogenous to the native polynucleotide containing the coding sequence to be expressed. Additionally, the promoter is generally heterologous with respect to the recombinant vector sequences within which the construct promoter/coding sequence has been inserted.
  • Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKR232-8 and pCM7 vectors.
  • CAT chloramphenicol transferase
  • Preferred bacterial promoters are the Lad, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the gpt, lambda PR, PL and frp promoters (EP 0036776), the polyhedrin promoter, or the plO protein promoter from baculovirus (Kit Novagen) (Smith et al., (1983) Mol Cell Biol 3(12):2156-65; O'Reilly et al., 1992), the lambda PR promoter or also the trc promoter.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late
  • promoters specific for a particular cell type may be chosen, such as those facilitating expression in adipose tissue, muscle tissue, or liver. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art. The choice of a promoter is well within the ability of a person skilled in the field of genetic engineering. For example, one may refer to Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989), or also to the procedures described by Fuller et al. (1996) Immunology in Current Protocols in Molecular Biology.
  • Bacterial vectors As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and a bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, but are not limited to, pRR223-3 (Pharmacia, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA).
  • Baculovirus vectors A suitable vector for the expression of polypeptides of the invention is a baculovirus vector that can be propagated in insect cells and in insect cell lines.
  • a specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC N°CRL 1711) which is derived from Spodoptera frugiperda.
  • mammalian vectors Further suitable vectors for the expression of polypeptides of the invention are mammalian vectors.
  • a number of suitable vector systems are known to those skilled in the art, for example, pcDNA4HisMax, pcDNA3.1Hygro-His andpcDNA3.1Hygro.
  • Viral vectors In one specific embodiment, the vector is derived from an adenovirus. Prefe ⁇ ed adenovirus vectors according to the invention are those described by Feldman and Steg (1996; Semin Interv
  • adenovirus type 2 or 5 Ad 2 or Ad 5
  • Ad 2 or Ad 5 adenovirus of animal origin
  • Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery systems of choice for the transfer of exogenous polynucleotides in vivo, particularly to mammals, including humans.
  • Particularly prefe ⁇ ed Murine Leukemia Vimses include the 4070 A and the 1504A vimses, Abelson (ATCC No VR-999), Friend (ATCC No VR-245), Gross (ATCC No VR-590), Rauscher (ATCC No VR-998) and Moloney Murine Leukemia Vims (ATCC No VR-190; PCT Application No WO 94/24298).
  • Particularly prefe ⁇ ed Rous Sarcoma Vimses include Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728).
  • Other prefe ⁇ ed retroviral vectors are those described in Roth et al.
  • AAV adeno- associated vims
  • the adeno-associated vims is a naturally occurring defective virus that requires another vims, such as an adenovirus or a he ⁇ es virus, as a helper vims for efficient replication and a productive life cycle (Muzyczka et al., (1992) Curr Top Microbiol Immunol 158:97-129).
  • these constructs In order to effect expression of the polynucleotides of the invention, these constructs must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cell lines, or in vivo or ex vivo, as in the treatment of certain disease states.
  • non-viral methods for the transfer of polynucleotides into cultured mammalian cells include, without being limited to, calcium phosphate precipitation (Graham et al., (1973) Virology 54(2):536-9; Chen et al., (1987) Mol Cell Biol 7(8):2745-52), DEAE-dexfran (Gopal, (1985) Mol Cell Biol 5(5):1188-90), electroporation (Tur-Raspa et al., (1986) Mol Cell Biol 6(2):716-8; Potter et al., (1984) Proc Natl Acad Sci USA 81(22):7161-5.), direct microinjection (Harland et al., (1985) J Cell Biol 101(3):1094-9), DNA- loaded liposomes (Nicolau et al., (1982) Biochim Biophys Acta 721(2): 185-90; Fraley et al., (1979) Pro
  • One specific embodiment for a method for delivering a protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable ca ⁇ ier and a naked polynucleotide operatively coding for the polypeptide of interest into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect.
  • This is particularly applicable for transfer in vitro but it may be applied to in vivo as well.
  • the transfer of a naked polynucleotide of the invention, including a polynucleotide construct of the invention, into cells may be proceeded with a particle bombardment (biolistic), said particles being DNA-coated microprojectiles accelerated to a high velocity allowing them to pierce cell membranes and enter cells without killing them, such as described by Klein et al. ((1990) Cu ⁇ Genet Feb; 17(2):97-103).
  • a particle bombardment biolistic
  • the polynucleotide of the invention may be entrapped in a liposome (Ghosh and Bacchawat, (1991) Targeted Diagn Ther 4:87-103; Wong et al., (1980) Gene
  • the invention provides a composition for the in vivo production of GMG-1 globular head polypeptide described herein. It comprises a naked polynucleotide operatively coding for this polypeptide, in solution in a physiologically acceptable ca ⁇ ier, and suitable for introduction into a tissue to cause cells of the tissue to express the said polypeptide.
  • the amount of vector to be injected to the desired host organism varies according to the site of injection. As an indicative dose, it will be injected between 0.1 and 100 ⁇ g of the vector in an animal body, preferably a mammal body, for example a mouse body.
  • the vector according to the invention may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell.
  • a somatic cell such as a muscle cell.
  • the cell that has been transformed with the vector coding for the desired GMG-1 globular head polypeptide or the desired fragment thereof is reinfroduced into the animal body in order to deliver the recombinant protein within the body either locally or systemically.
  • Another object of the invention consists of host cells recombinant for, i.e., that have been transformed or transfected with one of the polynucleotides described herein, and more precisely a polynucleotide comprising a polynucleotide encoding an GMG-1 polypeptide fragment of the invention such as any one of those described in "Polynucleotides of the Invention". These polynucleotides can be present in cells as a result of transient or stable transfection.
  • the invention includes host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as any one of those described in "Recombinant Vectors of the Invention".
  • a recombinant host cell of the invention comprises at least one of the polynucleotides or the recombinant vectors of the invention that are described herein.
  • Prefe ⁇ ed host cells used as recipients for the recombinant vectors of the invention are the following : a) Prokaryotic host cells : Escherichia coli strains (I.E. DH5- ⁇ strain), Bacillus subtilis,
  • Eukaryotic host cells HeLa cells (ATCC N°CCL2; N°CCL2.1; N°CCL2.2), Cv 1 cells (ATCC N°CCL70), COS cells (ATCC N°CRL1650; N°CRL1651), Sf-9 cells (ATCC N°CRL1711), C127 cells (ATCC N° CRL-1804), 3T3 (ATCC N° CRL-6361), CHO (ATCC N° CCL-61), human kidney 293 (ATCC N° 45504; N° CRL-1573), BHK (ECACC N° 84100501; N° 84111301), PLC cells, HepG2, and Hep3B.
  • the constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting cmde extract retained for further purification.
  • Microbial cells employed in the expression of proteins can be dismpted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known by the skilled artisan.
  • these recombinant cells can be created in vitro or in vivo in an animal, preferably a mammal, most preferably selected from the group consisting of mice, rats, dogs, pigs, sheep, cattle, and primates, not to include humans.
  • Recombinant cells created in vitro can also be later surgically implanted in an animal, for example. Methods to create recombinant cells in vivo in animals are well known in the art.
  • compositions may be produced, and methods performed, by techniques known in the art, such as those described in U.S.
  • the GMG-1 gene expression in mammalian, and typically human, cells may be rendered defective, or alternatively it may be enhanced, with the insertion of an GMG-1 genomic or cDNA sequence with the replacement of the GMG-1 gene counte ⁇ art in the genome of an animal cell by an GMG-1 polynucleotide according to the invention.
  • These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.
  • One kind of host cell that may be used are mammalian zygotes, such as murine zygotes.
  • EDTA containing 100 mM NaCl, 30 ⁇ M spermine, and 70 ⁇ M spermidine.
  • polyamines and high salt concenfrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al ((1993) Nature 362(6417):258-61).
  • Any one of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line.
  • ES cell lines are derived from pluripotent, uncommitted cells of the inner cell mass of pre-implantation blastocysts.
  • the constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the present invention also provides methods and compositions for the generation of non- human animals and plants that express recombinant GMG-1 polypeptide, i.e. recombinant gGMG-1 polypeptide fragment or full-length GMG-1 polypeptide.
  • the animals or plants can be transgenic, i.e. each of their cells contains a gene encoding the GMG-1 polypeptide, or, alternatively, a polynucleotide encoding the polypeptide can be introduced into somatic cells of the animal or plant, e.g. into mammary secretory epithelial cells of a mammal.
  • the non- human animal is a mammal such as a cow, sheep, goat, pig, or rabbit.
  • said gene encoding said gGMG-1 polypeptide fragment or said full-length GMG-1 polypeptide comprises the polynucleotide of SEQ ID NO : 5.
  • transgenic mammals can be produced, e.g., by fransfecting a pluripotential stem cell such as an ES cell with a polynucleotide encoding a polypeptide of interest. Successfully fransformed ES cells can then be introduced into an early stage embryo that is then implanted into the utems of a mammal of the same species.
  • a pluripotential stem cell such as an ES cell with a polynucleotide encoding a polypeptide of interest.
  • Successfully fransformed ES cells can then be introduced into an early stage embryo that is then implanted into the utems of a mammal of the same species.
  • transgenic mammals are described, e.g., in Wall et al. (1992) J Cell Biochem 199249(2): 113-20; Hogan, et al. (1986) in Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; in WO 91/08216; or in US Patent Number 4,736,866.
  • transgenic mammals are generated that secrete recombinant GMG-1 polypeptides in their milk.
  • the mammary gland is a highly efficient protein-producing organ, such methods can be used to produce protein concenfrations in the gram per liter range, and often significantly more.
  • expression in the mammary gland is accomplished by operably linking the polynucleotide encoding the GMG-1 polypeptide to a mammary gland specific promoter and, optionally, other regulatory elements.
  • Suitable promoters and other elements include, but are not limited to, those derived from mammalian short and long WAP, alpha, beta, and kappa, casein, alpha and beta lactoglobulin, beta-CN 5' genes, as well as the mouse mammary tumor vims (MMTV) promoter.
  • Such promoters and other elements may be derived from any mammal, including, but not limited to, cows, goats, sheep, pigs, mice, rabbits, and guinea pigs.
  • Promoter and other regulatory sequences, vectors, and other relevant teachings are provided, e.g., by Clark (1998) J Mammary Gland Biol Neoplasia 3:337-50; Jost et al.
  • the polypeptides of the invention can be produced in milk by introducing polynucleotides encoding the polypeptides into somatic cells of the mammary gland in vivo, e.g. mammary secreting epithelial cells.
  • plasmid DNA can be infused through the nipple canal, e.g. in association with DEAE-dextran (see, e.g., Hens et al. (2000) Biochim. Biophys. Acta 1523: 161-171), in association with a Iigand that can lead to receptor-mediated endocytosis of the construct (see, e.g., Sobolev et al. (1998) 273:7928-33), or in a viral vector such as a retroviral vector, e.g. the Gibbon ape leukemia vims (see, e.g., Archer et al. (1994) PNAS
  • the polynucleotide may be operably linked to a mammary gland specific promoter, as described above, or, alternatively, any strongly expressing promoter such as CMV or MoMLV LTR.
  • a mammary gland specific promoter as described above, or, alternatively, any strongly expressing promoter such as CMV or MoMLV LTR.
  • the suitability of any vector, promoter, regulatory element, etc. for use in the present invention can be assessed beforehand by fransfecting cells such as mammary epithelial cells, e.g.
  • the polynucleotides can be administered in any suitable formulation, at any of a range of concentrations (e.g. 1-500 ⁇ g/ml, preferably 50-100 ⁇ g/ml), at any volume (e.g. 1-100 ml, preferably 1 to 20 ml), and can be administered any number of times (e.g. 1, 2, 3, 5, or 10 times), at any frequency (e.g. every 1, 2, 3, 5, 10, or any number of days).
  • concentrations, frequencies, modes of administration, etc. will depend upon the particular polynucleotide, vector, animal, etc., and can readily be determined by one of skill in the art.
  • the quantity of milk obtained, and thus the quantity of GMG-1 polypeptides produced can be enhanced using any standard method of lacation induction, e.g. using hexesfrol, estrogen, and/or progesterone.
  • the polynucleotides used in such embodiments can either encode a full-length GMG-1 polypeptide or a gGMG-1 polypeptide fragment.
  • the encoded polypeptide will include a signal sequence to ensure the secretion of the protein into the milk.
  • the full-length protein can, e.g., be isolated from milk and cleaved in vitro using a suitable protease.
  • a second, protease-encoding polynucleotide can be introduced into the animal or into the mammary gland cells, whereby expression of the protease results in the cleavage of the GMG-1 polypeptide in vivo, thereby allowing the direct isolation of gGMG-1 polypeptide fragments from milk.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be freated by compounds of the invention include hyperlipidemia and hyperuricemia.
  • Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, ATDS- related weight loss, cancer-related weight loss, anorexia, and bulimia.
  • the gGMG-1 polypeptide fragments may also be used to enhance physical performance during work or exercise or enhance a feeling of general well-being. Physical performance activities include walking, running, jumping, lifting and/or climbing.
  • the gGMG-1 polypeptide fragments or antagonists thereof may also be used to treat dyslexia, attention-deficit disorder (ADD), attention-deficit hyperactivity disorder (ADHD), and psychiatric disorders such as schizophrenia by modulating fatty acid metabolism, more specifically, the production of certain long-chain polyunsaturated fatty acids.
  • ADD attention-deficit disorder
  • ADHD attention-deficit hyperactivity disorder
  • psychiatric disorders such as schizophrenia by modulating fatty acid metabolism, more specifically, the production of certain long-chain polyunsaturated fatty acids.
  • inventions relate to methods for the prophylaxis or treatment of inflammation-related disorders, such as atherosclerosis, comprising administering to a subject in need of freatment (alternatively on a timed daily basis) homotrimeric gGMG-1 polypeptide fragment (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce proinflammatory cytokines, and/or (ii) cell adhesion molecule levels in said animal or human subject.
  • freatment alternatively on a timed daily basis
  • homotrimeric gGMG-1 polypeptide fragment or polynucleotide encoding said polypeptide
  • the gGMG-1 polypeptide fragments of the invention may be provided alone or in combination with other pharmaceutically or physiologically acceptable compounds.
  • Other compounds useful for the treatment of obesity and other diseases and disorders are cu ⁇ ently well-known in the art.
  • the gGMG-1 polypeptide fragments are useful for, and used in, the freatment of insulin resistance and diabetes using methods described herein and known in the art.
  • a prefe ⁇ ed embodiments relates to process for the therapeutic modification and regulation of glucose metabolism in an animal or human subject, which comprises administering to a subject in need of freatment (alternatively on a timed daily basis) an OBG or GMG-1 polypeptide fragment (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.
  • inventions relate to methods for the prophylaxis or treatment of diabetes comprising administering to a subject in need of freatment (alternatively on a timed daily basis) an OBG or GMG-1 polypeptide fragment (or polynucleotide encoding said polypeptide) in dosage amount and for a period sufficient to reduce plasma glucose levels in said animal or human subject.
  • Suitable routes of administration include oral, nasal, rectal, transmucosal, or intestinal administration, parenteral delivery, including intramuscular, subcutaneous, inframedullary injections, as well as infrathecal, direct infraventricular, intravenous, intraperitoneal, infranasal, infrapulmonary
  • compositions and medicaments for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries. Proper formulation is dependent upon the route of administration chosen.
  • compositions that can be taken orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable gaseous propellant, e.g., carbon dioxide.
  • a suitable gaseous propellant e.g., carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin, for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical or physiologically acceptable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form.
  • Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder or lyophilized form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.
  • a suitable vehicle such as sterile pyrogen-free water
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by inframuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained release materials have been established and are well known by those skilled in the art.
  • Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve their intended pu ⁇ ose. More specifically, a therapeutically effective amount means an amount effective to prevent development of or to alleviate the existing symptoms of the subject being treated. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes or encompasses a concentration point or range shown to increase leptin or lipoprotein uptake or binding in an in vitro system. Such information can be used to more accurately determine useful doses in humans.
  • a therapeutically effective dose refers to that amount of the compound that results in amelioration of symptoms in a patient. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50, (the dose lethal to 50% of the test population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. Compounds that exhibit high therapeutic indices are prefe ⁇ ed.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50, with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1).
  • Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain or prevent weight loss or gain, depending on the particular situation. Dosages necessary to achieve these effects will depend on individual characteristics and route of administration.
  • Dosage intervals can also be determined using the value for the minimum effective concentration.
  • Compounds should be administered using a regimen that maintains plasma levels above the minimum effective concentration for 10-90% of the time, preferably between 30-90%; and most preferably between 50-90%. hi cases of local administration or selective uptake, the effective local concenfration of the drug may not be related to plasma concentration.
  • composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician.
  • a prefe ⁇ ed dosage range for the amount of an GMG-1 polypeptide fragment of the invention which can be administered on a daily or regular basis to achieve desired results, including a reduction in levels of circulating plasma friglyceride-rich lipoproteins, range from 0.01 - 0.5 mg/kg body mass.
  • a more prefe ⁇ ed dosage range is from 0.05 - 0.1 mg/kg.
  • these daily dosages can be delivered or administered in small amounts periodically during the course of a day. It is noted that these dosage ranges are only prefe ⁇ ed ranges and are not meant to be limiting to the invention.
  • mice with gGMG-1 polypeptide fragments results in decreased triglyceride levels, decreased free fatty acid levels, decreased glucose levels, and decreased body weight as well as increased muscle oxidation.
  • the invention is drawn inter alia to methods of preventing or treating obesity-related diseases and disorders comprising providing an individual in need of such treatment with a gGMG-1 polypeptide fragment of the invention.
  • the GMG-1 polypeptide fragment has obesity-related activity either in vitro or in vivo.
  • the GMG-1 polypeptide fragment is provided to the individual in a pharmaceutical composition that is preferably taken orally.
  • the individual is a mammal, and most preferably a human, hi prefe ⁇ ed embodiments, the obesity-related disease or disorder is selected from the group consisting of atherosclerosis, cardiovascular disease, impaired glucose tolerance, insulin resistance, hypertension, stroke, Syndrome X, Type I diabetes, Type II diabetes and lipoafrophic diabetes.
  • Diabetes-related complications to be freated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity- related disorders to be freated by compounds of the invention include hyperlipidemia, hypertriglyceridemia, and hyperuricemia.
  • Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, neoplasia-related weight loss, anorexia, and bulimia.
  • GMG-1 polypeptide polypeptide fragments in pharmaceutical compositions are used to modulate body weight in healthy individuals for cosmetic reasons.
  • the invention also features a method of preventing or treating obesity-related diseases and disorders comprising providing an individual in need of such treatment with a compound identified by assays of the invention (described in Section VI of the Prefe ⁇ ed Embodiments of the Invention and in the Examples).
  • a compound identified by assays of the invention (described in Section VI of the Prefe ⁇ ed Embodiments of the Invention and in the Examples).
  • these compounds antagonize or agonize effects of gGMG-1 polypeptide fragments in cells in vitro, muscles ex vivo, or in animal models.
  • these compounds agonize or antagonize the effects of gGMG-1 polypeptide fragments on leptin and/or lipoprotein uptake and/or binding.
  • these compounds prevent the interaction, binding, or uptake of gGMG-1 polypeptide fragments with LSR in vitro or in vivo.
  • the compound is provided to the individual in a pharmaceutical composition that is preferably taken orally.
  • the individual is a mammal, and most preferably a human.
  • the obesity-related disease or disorder is selected from the group consisting of obesity and obesity- related diseases and disorders such as atherosclerosis, heart disease, impaired glucose tolerance, insulin resistance, hypertension, stroke, Syndrome X, Type I diabetes, Type TI diabetes, and lipoatrophic diabetes.
  • Diabetes-related complications to be freated by the methods of the invention include microangiopathic lesions, ocular lesions, retinopathy, neuropathy and renal lesions.
  • Heart disease includes, but is not limited to, cardiac insufficiency, coronary insufficiency, and high blood pressure.
  • Other obesity-related disorders to be treated by compounds of the invention include hyperlipidemia, hypertriglyceridemia, and hyperuricemia.
  • Yet other obesity-related diseases or disorders of the invention include cachexia, wasting, AIDS-related weight loss, neoplasia-related weight loss, anorexia, and bulimia.
  • the pharmaceutical compositions are used to modulate body weight for cosmetic reasons.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to confrol blood glucose in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, in combination with insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to treat inflammatory disorders, including, but not limited to, atherosclerosis, coronary heart disease, myocardial infarction, and disseminated intravascular coagulation.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to confrol body weight in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, in combination with insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to confrol blood glucose in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, alone, without combination of insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to confrol body weight in some individuals, particularly those with Type II diabetes or insulin resistance, alone, without combination of insulin therapy.
  • the present invention may be used in complementary therapy, particularly in some individuals, particularly those with Type I diabetes, Type TI diabetes, or insulin resistance, to improve their weight or glucose confrol in combination with an oral insulin secretagogue or an insulin sensitising agent.
  • the oral insulin secretagogue is 1,1- dimethyl-2-(2-mo ⁇ holino phenyl)guanidine fumarate (BTS67582) or a sulphonylurea selected from tolbutamide, tolazamide, chlo ⁇ ropamide, glibenclamide, glimepiride, glipizide and glidazide.
  • the insulin sensitising agent is selected from metformin, ciglitazone, troglitazone and pioglitazone.
  • the present invention further provides a method of improving the body weight or glucose control of some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, alone, without an oral insulin secretagogue or an insulin sensitising agent.
  • the present invention may be administered either concomitantly or concu ⁇ ently, with the oral insulin secretagogue or insulin sensitising agent for example in the form of separate dosage units to be used simultaneously, separately or sequentially
  • the present invention further provides for a composition of pharmaceutical or physiologically acceptable composition and an oral insulin secretagogue or insulin sensitising agent as a combined preparation for simultaneous, separate or sequential use for the improvement of body weight or glucose confrol in some individuals, particularly those with Type I diabetes, Type ⁇ diabetes, or insulin resistance.
  • the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an insulin sensitiser.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some individuals, particularly those with Type I diabetes, Type II diabetes, or insulin resistance, in combination with insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition can be used as a method to improve insulin sensitivity in some individuals, particularly those with Type ⁇ diabetes or insulin resistance, without insulin therapy.
  • the present invention of said pharmaceutical or physiologically acceptable composition further provides a method for the use as an inhibitor of the progression from impaired glucose tolerance to insulin resistance.
  • the instant invention is drawn to treatment with gGMG-1 polypeptide fragments where an individual is shown to have a particular genotype for an GMG-1 marker, or where they have been shown to have a reduced amount of plasma GMG-1, either full-length or preferably a more biologically active fragment of GMG-1, as compared to control values, e.g. values representative of non-diseased individuals, or as compared to that individual prior to the onset of a disease or condition, i either case, treatment comprises providing pharmaceutically acceptable gGMG-1 or GMG-1 polypeptide fragments to the individual.
  • the exact amount of gGMG-1 fragment provided would be determined through clinical trials under the guidance of qualified physicians, but would be expected to be in the range of 5-7 mg per individual per day.
  • a prefe ⁇ ed range would be from 0.5 to 14 mg per individual per day, with a highly prefe ⁇ ed range being between 1 and 10 mg per individual per day.
  • Individuals who could benefit from treatment with gGMG-1 or GMG-1 polypeptide fragments could be identified through at least two methods: plasma serum level determinations and genotyping.
  • GMG-1 levels Preliminary studies have shown that obese people have lower levels of full-length GMG-1 than non- obese people.
  • the invention preferably is drawn to freatment of individuals with low levels of the biologically active fragment of GMG-1 with gGMG-1 polypeptide fragments of the invention
  • GMG-1 or GMG-1 polypeptide fragments of the present invention are administered to individuals, preferably obese individuals, that levels of full-length GMG-1 (or alternatively a mature GMG-1 polypeptide fragment) at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, about 100% or 100% lower than non-obese individuals, preferably healthy individuals as determined by a physician using normal standards in the art.
  • Methods to determine and compare the levels of full-length GMG-1 in individuals are well-known in the art and include, but are not limited to using an antibody specific for GMG-1 in a format such as a Radio Immune Assay, ELISA, Western blot, dotblot, or as part of an a ⁇ ay, for example.
  • Methods of generating antibodies to, and detection of, GMG-1 and fragments thereof as well as to proteins with SNPs are included in the present invention and are discussed in PCT/TB99/01858, US application No. 09/434,848, and WO 99/07736, hereby inco ⁇ orated herein by reference in its entirety including and drawings, figures, or tables. Further, antibodies specific for GMG-l/gGMG-1 polypeptide fragments of the invention, their generation, and their use are described herein. VII. Assays for Identifying Modulators of GMG-1 Polypeptide Fragment Activity
  • the invention features methods of screening for one or more compounds that modulate gGMG-1 polypeptide fragment activity in cells, that includes providing potential compounds to be tested to the cells, and where modulation of a gGMG-1 polypeptide fragment effect or activity indicates the one or more compounds.
  • Exemplary assays that may be used are described in the
  • assays in which an effect is observed based on the addition of gGMG-1 polypeptide fragment can also be used to screen for modulators of gGMG-1 polypeptide fragment activity or effects of the presence of gGMG-1 polypeptide fragment on cells.
  • the essential step is to apply an unknown compound and then to monitor an assay for a change from what is seen when only gGMG-1 polypeptide fragment is applied to the cell.
  • a change is defined as something that is significantly different in the presence of the compound plus gGMG-1 polypeptide fragment compared to gGMG- 1 polypeptide fragment alone, hi this case, significantly different would be an "increase” or a "decrease” in a measurable effect of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%.
  • modulation refers to a measurable change in an activity. Examples include, but are not limited to, lipolysis stimulated receptor (LSR) modulation, leptin modulation, lipoprotein modulation, plasma FFA levels, FFA oxidation, TG levels, glucose levels, and weight. These effects can be in vitro or preferably in vivo. Modulation of an activity can be either an increase or a decrease in the activity. Thus, LSR activity can be increased or decreased, leptin activity can be increased or decreased, and lipoprotein activity can be increased or decreased. Similarly, FFA, TG, and glucose levels (and weight) can be increased or decreased in vivo Free Fatty Acid oxidation can be increased or decreased in vivo or ex vivo.
  • LSR lipolysis stimulated receptor
  • leptin modulation lipoprotein modulation
  • plasma FFA levels FFA oxidation
  • FFA oxidation TG levels
  • glucose levels and weight
  • LSR activity is meant expression of LSR on the surface of the cell, or in a particular conformation, as well as its ability to bind, uptake, and degrade leptin and lipoprotein.
  • leptin activity is meant its binding, uptake and degradation by LSR, as well as its transport across a blood brain barrier, and potentially these occu ⁇ ences where LSR is not necessarily the mediating factor or the only mediating factor.
  • lipoprotein activity is meant its binding, uptake and degradation by LSR, as well as these occu ⁇ ences where LSR is not necessarily the mediating factor or the only mediating factor. Exemplary assays are provided in Example 4-5, 7-14, 16, and 18.
  • assay and other comparable assays can be used to determine/identify compounds that modulate gGMG-1 polypeptide fragment activity. In some cases it may be important to identify compounds that modulate some but not all of the gGMG-1 polypeptide fragment activities, although preferably all activities are modified.
  • increasing refers to the ability of a compound to increase an gGMG-1 polypeptide fragment activity in some measurable way compared to the effect of an gGMG-1 polypeptide fragment in its absence.
  • an increase in activity is at least 25%, 30%, 35%,
  • decreasing refers to the ability of a compound to decrease an activity in some measurable way compared to the effect of a gGMG-1 fragment in its absence. For example, the presence of the compound decreases the plasma concenfrations of FFA,
  • TG TG
  • glucose in mice.
  • a decrease in activity is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% as compared to the level of activity in the presence of the gGMG-1 polypeptide fragment alone.
  • the invention features a method for identifying a potential compound to modulate body mass in individuals in need of modulating body mass comprising: a) contacting a cell with a gGMG-1 polypeptide fragment and a candidate compound; b) detecting a result selected from the group consisting of LSR modulation, leptin modulation, lipoprotein modulation, FFA oxidation modulation; and c) wherein said result identifies said potential compound if said result differs from said result when said cell is contacted with the gGMG-1 polypeptide fragment alone.
  • said contacting further comprises a Iigand of said LSR.
  • said Iigand is selected from the group consisting of cytokine, lipoprotein, free fatty acids, and Clq, and more preferably said cytokine is leptin, and most preferably said leptin is a leptin polypeptide fragment as described in US Provisional application No. 60/155,506 hereby inco ⁇ orated by reference herein in its entirety including any figures, drawings, or tables. hi other prefe ⁇ ed embodiments, said gGMG-1 polypeptide fragment is mouse or is human.
  • said cell is selected from the group consisting of PLC, CHO-K1, Hep3B, and HepG2.
  • said lipoprotein modulation is selected from the group consisting of binding, uptake, and degradation.
  • said modulation is an increase in said binding, uptake, or degradation.
  • said modulation is a decrease in said binding, uptake, or degradation.
  • leptin modulation is selected from the group consisting of binding, uptake, degradation, and transport.
  • said modulation is an increase in said binding, uptake, degradation, or transport.
  • said modulation is a decrease in said binding, uptake, degradation, or fransport.
  • said transport is across a blood-brain barrier.
  • said LSR modulation is expression on the surface of said cell.
  • said detecting comprises FACS, more preferably said detecting further comprises antibodies that bind specifically to said LSR, and most preferably said antibodies bind specifically to the carboxy terminus of said LSR.
  • said potential compound is selected from the group consisting of peptides, peptide libraries, non-peptide libraries, peptoids, fatty acids, lipoproteins, medicaments, antibodies, small molecules, and proteases.
  • a prefe ⁇ ed embodiment of the present invention is directed to eiptope-bearing polypeptides and epitope-bearing polypeptide fragments.
  • These epitopes may be "antigenic epitopes” or both an “antigenic epitope” and an “immunogenic epitope”.
  • An "immunogenic epitope” is defined as a part of a protein that elicits an antibody response in vivo when the polypeptide is the immunogen.
  • a region of polypeptide to which an antibody binds is defined as an "antigenic determinant" or "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes.
  • An epitope can comprise as few as 3 amino acids in a spatial conformation which is unique to the epitope. Generally an epitope consists of at least 6 such amino acids, and more often at least 8-10 such amino acids. In prefe ⁇ ed embodiment, antigenic epitopes comprise a number of amino acids that is any integer between 3 and 50. Fragments which function as epitopes may be produced by any conventional means. See, e.g., Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985), further described in U.S. Patent No. 4,631,211.
  • Methods for determining the amino acids which make up an immunogenic epitope include x-ray crystallography, 2-dimensional nuclear magnetic resonance, and epitope mapping, e.g., the Pepscan method described by H. Mario Geysen et al. (1984); Proc. Natl. Acad. Sci. U.S.A. 81:3998-4002; PCT Publication No. WO 84/03564; and PCT Publication No. WO 84/03506.
  • Another example is the algorithm of Jameson and Wolf, Comp. Appl. Biosci. 4:181-186 (1988) (said references inco ⁇ orated by reference in their entireties).
  • the Jameson-Wolf antigenic analysis for example, may be performed using the computer program PROTEAN, using default parameters (Version 4.0 Windows, DNASTAR, Inc., 1228 South Park Street Madison, WT).
  • the epitope-bearing fragments of the present invention preferably comprises 6 to 50 amino acids (i.e. any integer between 6 and 50, inclusive) of a polypeptide of the present invention. Also, included in the present invention are antigenic fragments between the integers of 6 and the full length sequence of the sequence listing. All combinations of sequences between the integers of 6 and the full-length sequence of a polypeptide of the present invention are included.
  • the epitope- bearing fragments may be specified by either the number of contiguous amino acid residues (as a sub-genus) or by specific N-terminal and C-terminal positions (as species) as described above for the polypeptide fragments of the present invention. Any number of epitope-bearing fragments of the present invention may also be excluded in the same manner.
  • Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies that specifically bind the epitope (See,Wilson et al., 1984; and Sutcliffe, J. G. et al., 1983). The antibodies are then used in various techniques such as diagnostic and tissue/cell identification techniques, as described herein, and in purification methods.
  • immunogenic epitopes can be used to induce antibodies according to methods well known in the art (See, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. et al.;(1985) and Bittle, F. J. et al., (1985).
  • a prefe ⁇ ed immunogenic epitope includes the polypeptides of the sequence listing.
  • the immunogenic epitopes may be presented together with a ca ⁇ ier protein, such as an albumin, to an animal system (such as rabbit or mouse) if necessary.
  • Immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting.).
  • Epitope-bearing polypeptides of the present invention are used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods (See, e.g., Sutcliffe, et al., supra; Wilson, et al., supra, and Bittle, et al., 1985).
  • animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (RLH) or tetanus toxoid.
  • a macromolecular carrier such as keyhole limpet hemacyanin (RLH) or tetanus toxoid.
  • RH keyhole limpet hemacyanin
  • peptides containing cysteine residues may be coupled to a ca ⁇ ier using a linker such as - maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to ca ⁇ iers using a more general linking agent such as glutaraldehyde.
  • Animals such as rabbits, rats and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or infradermal injection of emulsions containing about 100 ⁇ gs of peptide or carrier protein and Freund's adjuvant.
  • emulsions containing about 100 ⁇ gs of peptide or carrier protein and Freund's adjuvant.
  • booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody, which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adso ⁇ tion to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • the polypeptides of the present invention including, but not limited to, polypeptides comprising an immunogenic or antigenic epitope can be fused to heterologous polypeptide sequences.
  • polypeptides of the present invention may be fused with the constant region comprising portions of immunoglobulins (IgA, IgE, IgG, IgM), or portions of the constant region (CHI, CH2, CH3, any combination thereof including both entire domains and portions thereof) resulting in chimeric polypeptides.
  • IgA, IgE, IgG, IgM immunoglobulins
  • CHI, CH2, CH3, any combination thereof including both entire domains and portions thereof resulting in chimeric polypeptides.
  • CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins See, e.g., EPA 0,394,827; and Traunecker et al., 1988).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion can also be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone (See, e.g., Fountoulakis et al., 1995).
  • Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag to aid in detection and purification of the expressed polypeptide.
  • DNA shuffling may be employed to modulate the activities of polypeptides of the present invention thereby effectively generating agonists and antagonists of the polypeptides. See, for example, U.S. Patent Nos.: 5,605,793; 5,811,238; 5,834,252; 5,837,458; and Patten, P.A., et al., (1997); Harayama, S., (1998); Hansson, L.O., et al (1999); and Lorenzo, M.M.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of coding polynucleotides of the invention, or the polypeptides encoded thereby may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the present invention further relates to antibodies and T-cell antigen receptors (TCR), which specifically bind the polypeptides, and more specifically, the epitopes of the polypeptides of the present invention.
  • TCR T-cell antigen receptors
  • the antibodies of the present invention include IgG (including IgG 1, IgG2, IgG3, and IgG4), IgA (including IgAl and IgA2), IgD, IgE, or IgM, and IgY.
  • antibody is meant to include whole antibodies, including single-chain whole antibodies, and antigen binding fragments thereof.
  • the antibodies are human antigen binding antibody fragments of the present invention include, but are not limited to, Fab, Fab' F(ab)2 and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a V L or V H domain.
  • the antibodies may be from any animal origin including birds and mammals.
  • the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken.
  • Antigen-binding antibody fragments may comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are any combinations of variable region(s) and hinge region, CHI, CH2, and CH3 domains.
  • the present invention further includes chimeric, humanized, and human monoclonal and polyclonal antibodies, which specifically bind the polypeptides of the present invention.
  • the present invention further includes antibodies that are anti-idiotypic to the antibodies of the present invention.
  • the antibodies of the present invention may be monospecific, bispecific, and trispecific or have greater multispecificity.
  • Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. (1991); US Patents 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; Rostelny, S.A. et al. (1992).
  • Antibodies of the present invention may be described or specified in terms of the epitope(s) or epitope-bearing portion(s) of a polypeptide of the present invention, which are recognized or specifically bound by the antibody, hi the case of proteins of the present invention secreted proteins, the antibodies may specifically bind a full-length protein encoded by a nucleic acid of the present invention, a mature protein (i.e., the protein generated by cleavage of the signal peptide) encoded by a nucleic acid of the present invention, a signal peptide encoded by a nucleic acid of the present invention, or any other polypeptide of the present invention.
  • the epitope(s) or epitope bearing polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or otherwise described herein.
  • Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded as individual species. Therefore, the present invention includes antibodies that specifically bind specified polypeptides of the present invention, and allows for the exclusion of the same.
  • Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not specifically bind any other analog, ortholog, or homolog of the polypeptides of the present invention are included. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein, eg., using FASTDB and the parameters set forth herein) to a polypeptide of the present invention are also included in the present invention.
  • antibodies which only bind polypeptides encoded by polynucleotides, which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein).
  • Antibodies of the present invention may also be described or specified in terms of their binding affinity.
  • Prefe ⁇ ed binding affinities include those with a dissociation constant or Rd value less than 5X10 "6 M, 10 "6 M, 5X10 “7 M, 10 '7 M, 5X10 “8 M, 10 “8 M, 5X10 "9 M, 10 "9 M, 5X10 " 10 M, 10 “10 M, 5X10 " ⁇ M, 10 " ⁇ M, 5X10 "12 M, 10 "12 M, 5X10 "13 M, 10 "13 M, 5X10 "14 M, 10 "14 M, 5X10 "15 M, and 10 '15 M.
  • Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples (See, e.g., Harlow et al., 1988).
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; US Patent 5,314,995; and EP 0 396 387.
  • the antibodies of the present invention may be prepared by any suitable method known in the art.
  • a polypeptide of the present invention or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • the term "monoclonal antibody” is not limited to antibodies produced through hybridoma technology.
  • the term "antibody” refers to a polypeptide or group of polypeptides which are comprised of at least one binding domain, where a binding domain is formed from the folding of variable domains of an antibody molecule to form three-dimensional binding spaces with an internal surface shape and charge distribution complementary to the features of an antigenic determinant of an antigen, which allows an immunological reaction with the antigen.
  • monoclonal antibody refers to an antibody that is derived from a single clone, including eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technology.
  • Fab and F(ab')2 fragments may be produced, for example, from hybridoma-produced antibodies by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • antibodies of the present invention can be produced through the application of recombinant DNA technology or through synthetic chemistry using methods known in the art.
  • the antibodies of the present invention can be prepared using various phage display methods known in the art. hi phage display methods, functional antibody domains are displayed on the surface of a phage particle, which ca ⁇ ies polynucleotide sequences encoding them. Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g. human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.
  • a repertoire or combinatorial antibody library e.g. human or murine
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene HI or gene VHI protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman U. et al. (1995); Ames, R.S. et al. (1995); Kettleborough, CA. et al. (1994); Persic, L. et al. (1997); Burton, D.R. et al. (1994);
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.
  • techniques to recombinantly produce Fab, Fab' F(ab)2 and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R.L. et al. (1992); and Sawai, H. et al. (1995); and Better, M. et al. (1988).
  • Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; US Patent 5,530,101; and 5,585,089), veneering or resurfacing, (EP 0 592 106; EP 0 519 596; Padlan E.A., 1991; Studnicka G.M. et al., 1994; Roguska M.A. et al., 1994), and chain shuffling (US Patent 5,565,332).
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above.
  • antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention may be specific for antigens other than polypeptides of the present invention.
  • antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vifro immunoassays and purification methods using methods known in the art (See e.g., Harbor et al.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion fused to a polypeptide of the present invention may comprise the hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
  • the polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half-life of the polypeptides or for use in immunoassays using methods known in the art.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.
  • Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM.
  • Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See e.g., US Patents 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al. (1991); Zheng, X.X. et al. (1995); and Vil, H. et al. (1992).
  • the invention further relates to antibodies that act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies that dis pt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. Included are both receptor-specific antibodies and ligand-specific antibodies. Included are receptor-specific antibodies, which do not prevent Iigand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. Also include are receptor-specific antibodies which both prevent Iigand binding and receptor activation.
  • neutralizing antibodies that bind the Iigand and prevent binding of the Iigand to the receptor, as well as antibodies that bind the Iigand, thereby preventing receptor activation, but do not prevent the Iigand from binding the receptor.
  • antibodies that activate the receptor may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation.
  • the antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.
  • the above antibody agonists can be made using methods known in the art. See e.g., WO 96/40281; US Patent 5,811,097; Deng, B. et al. (1998); Chen, Z. et al.
  • antibodies of the polypeptides of the invention can, in turn, be utilized to generate anti-idiotypic antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art (See, e.g. Greenspan and Bona (1989);and Nissinoff (1991).
  • “mimic” the polypeptide multimerization or binding domain and, as a consequence, bind to and neutralize polypeptide or its Iigand.
  • neutralization anti-idiotypic antibodies can be used to bind a polypeptide of the invention or to bind its ligands/receptors, and therby block its biological activity,
  • the invention also concerns a purified or isolated antibody capable of specifically binding to a mutated full length or mature polypeptide of the present invention or to a fragment or variant thereof comprising an epitope of the mutated polypeptide.
  • the present invention concerns an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids of a polypeptide of the present invention and including at least one of the amino acids which can be encoded by the trait causing mutations.
  • Non-human animals or mammals whether wild-type or transgenic, which express a different species of a polypeptide of the present invention than the one to which antibody binding is desired, and animals which do not express a polypeptide of the present invention (i.e. a knock out animal) are particularly useful for preparing antibodies.
  • Gene knock out animals will recognize all or most of the exposed regions of a polypeptide of the present invention as foreign antigens, and therefore produce antibodies with a wider a ⁇ ay of epitopes.
  • smaller polypeptides with only 10 to 30 amino acids may be useful in obtaining specific binding to any one of the polypeptides of the present invention.
  • the antibodies of the invention may be labeled by any one of the radioactive, fluorescent or enzymatic labels known in the art. Consequently, the invention is also directed to a method for detecting specifically the presence of a polypeptide of the present invention according to the invention in a biological sample, said method comprising the following steps: a) obtaining a biological sample suspected of containing a polypeptide of the present invention; b) contacting the biological sample with a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention under conditions suitable for antigen- antibody binding; and c) detecting the antigen-antibody complex formed.
  • the invention also concerns a diagnostic kit for detecting in vitro the presence of a polypeptide of the present invention in a biological sample, wherein said kit comprises: a) a polyclonal or monoclonal antibody that specifically binds a polypeptide of the present invention, optionally labeled; b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.
  • Monoclonal Antibody Production by Hybridoma Fusion Monoclonal antibody to epitopes of any of the peptides identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler, G. and Milstein, C, Nature 256:495 (1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein or peptides derived therefrom over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated.
  • the spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).
  • HAT media aminopterin
  • the successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
  • Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as Elisa, as originally described by Engvall, E., Meth. Enzymol. 70:419 (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use.
  • Still further particularly included are methods of selecting monoclonal antibodies, wherein said monoclonal antibody specifically binds to 134-285 but not to 136-285 of SEQ ID NO: 2 comprising the steps of: obtaining a sample comprising 134-285, obtaining a sample comprising 136-285, contacting said antibody with 134-285, contacting said antibody with 136-285, quantifying the level of antibody binding to 134-285 and to 136-285 by ELISA.
  • Still further particularly included are methods of selecting monoclonal antibodies, wherein said monoclonal antibody specifically binds to 47-217 but not to 49-217 of SEQ ID NO: 4 comprising the steps of: obtaining a sample comprising 47-217, obtaining a sample comprising 49-217, contacting said antibody with 47- 217, contacting said antibody with 49-217, quantifying the level of antibody binding to 47-217 and to 49-217 by ELISA.
  • Polyclonal antisemm containing antibodies to heterogenous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein or peptides derived therefrom described above, which can be unmodified or modified to enhance immunogenicity.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of ca ⁇ iers and adjuvant.
  • host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple infradermal sites appears to be most reliable.
  • An effective immunization protocol for rabbits can be found in Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991 (1971).
  • Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concenfrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al.,
  • Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980).
  • Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concenfrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample.
  • the antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.
  • the invention features methods of screening compounds for one or more antagonists of homotrimeric gGMG-1 polypeptide fragment activity, wherein said activity is selected from but not restricted to weight reduction, lipid partitioning, lipid metabolism, and insulin-like activity. Prefe ⁇ ed said compound is selected from but is not restricted to small molecular weight organic or inorganic compound, protein, peptide, carbohydrate, or lipid.
  • said gGMG-1 polypeptide fragment forming homofrimers having said activity is selected from amino acids 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-285, 7
  • said gGMG-1 polypeptide fragment forming homofrimers having said activity is selected from amino acids 45-285, 47-285, 126-285, 127-285, 132-285, 133- 285, or 134-285 of SEQ ID NO:2.
  • Other prefe ⁇ ed said gGMG-1 polypeptide fragment forming homofrimers having said activity is selected from amino acids 45-217, 47-217, or 88-217 of SEQ ID NO:4.
  • the invention further features methods of screening compounds for said antagonist of gGMG-1 polypeptide fragment activity comprising: a) contacting said homotrimeric gGMG-1 polypeptide fragment with or without said compound; b) detecting a result on the basis of activity, wherein said activity is selected from but not restricted to lipid partitioning, lipid metabolism, and insulin-like activity; and c) wherein said result identifies said compound as an antagonist of homotrimeric gGMG-1 polypeptide fragment activity if said result with compound differs from said result without compound.
  • Exemplary assays that may be used are described in Examples 4 and 18.
  • GMG-1 polypeptide used throughout the specification is intended to encompass the protein homologs mouse ACRP30 [Scherer, et al, "A novel se m protein similar to Clq, produced exclusively in adipocytes”; J Biol Chem 270, 26746- 26749 (1995)], mouse AdipoQ [Hu, et al, "AdipoQ is a novel adipose-specific gene dysregulated in obesity", J Biol Chem 271, 10697-10703 (1996)], human APM1 [Maeda, et al, "cDNA cloning and expression of a novel adipose specific collagen-like factor, APM1 (AdiPose Most abundant Gene transcript 1)", Biochem Biophys Res Commun 221, 286-289 (1996)] and human GBP28 [Nakano, et al, "Isolation and characterization of GBP28, a novel gelatin-binding protein purified from human plasma", J
  • EXAMPLE 1 Northern Analysis of GMG-1 DNA Analysis of GMG-1 expression in different human tissues (adult and fetal) and cell lines, as well as mouse embryos in different stages of development, is accomplished by using poly A + RNA blots purchased from Clontech (e.g. #7780-1, 7757-1, 7756-1, 7768-land 7763-1). Labeling of RNA probes is performed using the RNA Strip-EZ kit from Ambion as per manufacture's instructions. Hybridization of RNA probes to RNA blots is performed Ulfrahyb hybridization solution (Ambion).
  • blots are prehybridized for 30 min at 58°C (low-sfrigency) or 65°C (high stringency). After adding the labeled probe (2xl0 6 cpm/ml), blots are hybridized overnight (14-24 hrs), and washed 2 x 20 min at 50°C with 2x SSC/0.1% SDS (low stringency), 2 x 20 min at 58°C with lx SSC/0.1%SDS (medium stringency) and 2 x 20 min at 65°C with lx SSC/0.1%SDS (high stringency). After washings are completed blots are exposed on the phosphoimager (Molecular Dynamics) for 1-3 days.
  • GMG-1 polypeptides The activity of various preparations and various sequence variants of GMG-1 polypeptides are assessed using various in vitro assays including those provided below. These assays are also exemplary of those that can be used to develop GMG-1 polypeptide antagonists and agonists. To do that, the effect of GMG-1 polypeptides in the above assays, e.g. on leptin and/or LSR activity, in the presence of the candidate molecules would be compared with the effect of GMG-1 polypeptides in the assays in the absence of the candidate molecules. Since GMG-1 polypeptides are believed to reduce body weight in mice on a high-cafeteria diet (Example 5), these assays also serve to identify candidate treatments for reducing (or increasing) body weight.
  • Tests of efficacy of GMG-1 polypeptides on LSR can be performed using liver cell lines, including for example, PLC, HepG2, Hep3B (human), Hepa 1-6, BPRCL (mouse), or MCA-RH777, MCA-RH8994 (rat).
  • liver cell lines including for example, PLC, HepG2, Hep3B (human), Hepa 1-6, BPRCL (mouse), or MCA-RH777, MCA-RH8994 (rat).
  • BPRCL mouse liver cells (ATCC Repository) are plated at a density of 300,000 cells/well in 6-well plates (day 0) in DMEM (high glucose) containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992). Media is changed on day 2. On day 3, the confluent monolayers are washed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).
  • PBS phosphate-buffered saline
  • Cells are incubated at 37°C for 30 min with increasing concentrations of recombinant AdipoQ (AQ) or globular AdipoQ (AQ-GH) in DMEM containing 0.2% (w/v) BSA, 5 mM Hepes, 2 mM CaCl 2 , 3.7 g/L sodium bicarbonate, pH 7.5. Incubations are continued for 3 h at 37°C after addition of 10 ng/mL 125 I-mouse leptin (specific activity, 22100 cpm/ng). Monolayers are washed 2 times consecutively with PBS containing 0.2% BSA, followed by 1 wash with PBS/BSA, and then 2 times consecutively with PBS. Cells are lysed with 0.1 N NaOH containing 0.24 mM EDTA. Lysates are collected into tubes, and counted in a gamma-counter.
  • AQ recombinant AdipoQ
  • AQ-GH globular AdipoQ
  • the effect of GMG-1 polypeptides on leptin transport in the brain can be determined using brain-derived cells.
  • One method that is envisioned is to use the blood/brain barrier model described by Dehouck, et al (J Neurochem 54:1798-801, 1990; hereby inco ⁇ orated herein by reference in its entirety including any figures, tables, or drawings) that uses a co-culture of brain capillary endothelial cells and asfrocytes to test the effects of GMG-1 polypeptides on leptin (or other molecules) transport via LSR or other receptors.
  • This assay would be an indicator of the potential effect of GMG-1 polypeptides on leptin fransport to the brain and could be used to screen GMG-1 polypeptide variants for their ability to modulate leptin fransport through LSR or other receptors in the brain.
  • putative agonists and antagonists of the effect of GMG-1 polypeptides on leptin transport through LSR or other receptors could also be screened using this assay.
  • Increased fransport of leptin across the blood brain ba ⁇ ier would presumably increase its action as a satiety factor.
  • Flow cytometry is a laser-based technology that is used to measure characteristics of biological particles. The underlying principle of flow cytometry is that light is scattered and fluorescence is emitted as light from the excitation source strikes the moving particles.
  • Cells are pretreated with either intact GMG-1 polypeptides (or untreated) before harvesting and analysis by FACS.
  • Cells are harvested using non-enzymatic dissociation solution (Sigma), and then are incubated for 1 h at 4°C with a 1:200 dilution of anti-LSR 8 IB or an i ⁇ elevant anti-semm in PBS containing 1% (w/v) BSA. After washing twice with the same buffer, goat anti-rabbit FITC- conjugated antibody (Rockland, Gilbertsville, PA) is added to the cells, followed by a further incubation for 30 min at 4 °C. After washing, the cells are fixed in 2%> formalin. Flow cytometry analysis is done on a FACSCalibur cytometer (Becton-Dickinson, Franklin Lakes, NJ). Assay 2:
  • FACS buffer lx PBS/2% FBS, filter sterilized
  • the cell suspension is transfe ⁇ ed to a 15 mL conical tube and centrifuged at 1200 ⁇ m, 4°C for 5 minutes. Supernatant is discarded and cells are resuspended in 10 mL FACS buffer chilled to 4°C.
  • a cell count is performed and the cell density adjusted with FACS buffer to a concenfration of 1 x 10 6 cells/ mL.
  • One milliliter of cell suspension was added to each well of a 48 well plate for analysis. Cells are centrifuged at 1200 ⁇ m for 5 minutes at 4°C.
  • GMG-1 Polypeptides as Detected by Fluorescence Microscopy Fluorecein isothiocyanate (FITC) conjugation of GMG-1 polypeptides: Purified GMG-1 proteins at 1 mg/rnL concenfration are labeled with FITC using Sigma' s FluoroTag FITC conjugation kit (Stock No. FITC-1). Protocol outlined in the Sigma Handbook for small scale conjugation is followed for GMG-1 protein labeling.
  • Cell Culture C2C12 mouse skeletal muscle cells (ATCC, Manassas, VA CRL-1772) and
  • Hepa-1-6 mouse hepatocytes (ATCC, Manassas, VA CRL-1830) are seeded into 6 well plates at a cell density of 2xl0 5 cells per well. C2C12 and Hepa-1-6 cells are cultured according to repository's instructions for 24-48 hours prior to analysis. Assay is performed when cells were 80% confluent.
  • FITC labeled GMG-1 proteincellular binding and uptake using microscopy C2C12 and Hepa 1 -6 cells are incubated in the presence/absence of antibody directed against human LSR (8 IB : N-terminal sequence of human LSR; does not cross react with mouse LSR and 93 A: c-terminal sequence, cross reacts with mouse LSR) or an antisemm directed against gClqr (953) for 1 hour at 37°C, 5%> C02.
  • LSR antibodies are added to the media at a concenfration of 2 ⁇ g/mL.
  • the anti- gClqr antisemm is added to the media at a volume of 2.5 ⁇ L undiluted semm (high concenfration) or 1:100 dilution (low concenfration).
  • FITC-GMG-1 polypeptide 50 nM/mL is added to each cell culture well. Cells are again incubated for 1 hour at 37°C, 5% C02. Cells are washed 2x with PBS, cells are scraped from well into 1 mL of PBS. Cell suspension is transfe ⁇ ed to an eppendorf tube and centrifuged at 1000 ⁇ m for 2 minutes. Supernatant is removed and cells resuspended in 200 ⁇ L of PBS. Binding and uptake of FITC- GMG-1 polypeptide is analyzed by fluorescence microscopy under 40X magnification.
  • This assay may be useful for identifying agents that facilitate or prevent the uptake and/or binding of GMG-1 polypeptides to cells.
  • the effect of GMG-1 protein on the lipoprotein binding, internalizing and degrading activity of LSR can also be tested. Measurement of LSR as lipoprotein receptor is described in Bihain & Yen, ((1992) Biochemistry May 19;31(19):4628-36; hereby inco ⁇ orated herein in its entirety including any drawings, tables, or figures).
  • the effect of GMG-1 protein on the lipoprotein binding, internalizing and degrading activity of LSR (or other receptors) can be compared with that of intact GMG-1 protein, with untreated cells as an additional control. This assay can also be used to screen for active and inhibitory variants of GMG-1 protein, as well as agonists and antagonists of metabolic-related activity.
  • Human liver PLC cells (ATCC Repository) are plated at a density of 300,000 cells/well in 6- well plates (day 0) in DMEM (high glucose) containing glutamine and penicillin-streptomycin (Bihain & Yen, 1992). Media is changed on day 2. On day 3, the confluent monolayers are washed once with phosphate-buffered saline (PBS, pH 7.4) (2 mL/well).
  • PBS phosphate-buffered saline
  • the oleate-induced binding and uptake of LDL would be more affected by GMG-1 proteinas compared to the degradation.
  • This increased LSR activity would potentially result in an enhanced clearance of triglyceride-rich lipoproteins during the postprandial state.
  • more dietary fat would be removed through the liver, rather than being deposited in the adipose tissue.
  • This assay could be used to determine the efficiency of a compound (or agonists or antagonists) to increase or decrease LSR activity (or lipoprotein uptake, binding and degradation through other receptors), and thus affect the rate of clearance of triglyceride-rich lipoproteins.
  • C2C12 cells (murine skeletal muscle cell line; ATCC CRL 1772, Rockville, MD) are seeded sparsely (about 15-20%) in complete DMEM (w/glutamine, pen/strep, etc) + 10% FCS. Two days later they become 80-90% confluent. At this time, the media is changed to DMEM+2% horse semm to allow differentiation. The media is changed daily. Abundant myotube formation occurs after 3-4 days of being in 2% horse semm, although the exact time course of C2C12 differentiation depends on how long they have been passaged and how they have been maintained, among other things.
  • GMG-1 (1 to 2.5 ⁇ g/mL) is added the day after seeding when the cells are still in DMEM w/ 10% FCS. Two days after plating the cells (one day after GMG-1 was first added), at about 80-90% confluency, the media is changed to DMEM+2% horse semm plus GMG-1.
  • C2C12 cells are differentiated in the presence or absence of 2 ⁇ g/mL GMG-1 proteinfor 4 days.
  • oleate oxidation rates are determined by measuring conversion of l- 14 C-oleate (0.2 mM) to 14 C0 2 for 90 min. This experiment can be used to screen for active polypeptides and peptides as well as agonists and antagonists or activators and inhibitors of GMG-1 polypeptides.
  • the effect of GMG-1 on the rate of oleate oxidation can be compared in differentiated C2C12 cells (murine skeletal muscle cells; ATCC, Manassas, VA CRL-1772) and in a hepatocyte cell line (Hepal-6; ATCC, Manassas, VA CRL-1830). Cultured cells are maintained according to manufacturer's instructions. The oleate oxidation assay is performed as previously described
  • DMEM low serum differentiation media
  • Hepatocytes are kept in the same DMEM medium supplemented with 10% FCS for 2 days.
  • MEM preincubation media
  • cells are placed on ice. To determine triglyceride and protein content, cells are washed with 1 mL of lx PBS to remove residual media.
  • Analysis of samples is carried out on a Packard Spectra Count at a wavelength of 550 nm. Protein analysis is carried out on 25 ⁇ L of each supernatant sample using the BCA protein assay (Pierce) following manufacturer's instructions. Analysis of samples is ca ⁇ ied out on a Packard Specfra Count at a wavelength of 550 nm.
  • L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11. Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [ 3 H]-2-deoxyglucose (2DG) with or without GMG-1 polypeptide fragment in the presence or absence of insulin (10 "8 M) as has been previously described (Walker, P.S. et al. (1990) Glucose transport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription. JBC 265(3):1516-1523; and Rilp, A. et al. (1992) Stimulation of hexose transport by metformin in L6 muscle cells in culture.
  • Uptake of 2DG is expressed as the percentage change compared with confrol (no added insulin or GMG-1 polypeptide fragment). Values are presented as mean + SEM of sets of 4 wells per experiment. Differences between sets of wells are evaluated by Student's t test, probability values p ⁇ 0.05 are considered to be significant.
  • EXAMPLE 3 Effect of GMG-1 Polypeptides on Mice Fed a High-Fat Diet Experiments are performed using approximately 6 week old C57B1/6 mice (8 per group).
  • mice All mice are housed individually. The mice are maintained on a high fat diet throughout each experiment.
  • the high fat diet (cafeteria diet; D 12331 from Research Diets, Inc.) has the following composition: protein kcal%> 16, sucrose kcal%> 26, and fat kcal% 58.
  • the fat is primarily composed of coconut oil, hydrogenated.
  • mice After the mice are fed a high fat diet for 6 days, micro-osmotic pumps are inserted using isoflurane anesthesia, and are used to provide full-length GMG-1 polypeptides, GMG-1 polypeptide fragments, saline, and an i ⁇ elevant peptide to the mice subcutaneously (s.c.) for 18 days.
  • GMG-1 polypeptides are provided at doses of 100, 50, 25, and 2.5 ⁇ g/day and the i ⁇ elevant peptide is provided at 10 ⁇ g/day.
  • Body weight is measured on the first, third and fifth day of the high fat diet, and then daily after the start of freatment. Final blood samples are taken by cardiac puncture and are used to determine triglyceride (TG), total cholesterol (TC), glucose, leptin, and insulin levels. The amount of food consumed per day is also determined for each group.
  • TG triglyceride
  • TC total cholesterol
  • glucose leptin
  • insulin levels The amount of food consumed per
  • GMG-1 polypeptides preferably GMG-1 polypeptides comprising the Clq homology region, would be given in daily doses of about 6 mg protein per 70 kg person or about 10 mg per day. Other doses would also be tested, for instance 1 mg or 5 mg per day up to 20 mg, 50 mg, or 100 mg per day.
  • EXAMPLE 5 Tests of Metabolic-related Activity in a Murine Lipoafrophic Diabetes Model
  • leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodysfrophy (Shimomura et al. Nature 401 : 73-76 (1999); hereby inco ⁇ orated herein in its entirety including any drawings, figures, or tables).
  • Leptin was found to be less effective in a different lipodysfrophic mouse model of lipoafrophic diabetes (Grajova et al Nature 403: 850 (2000); hereby inco ⁇ orated herein in its entirety including any drawings, figures, or tables).
  • the instant invention encompasses the use of GMG-1 polypeptides for reducing the insulin resistance and hyperglycaemia in this model either alone or in combination with leptin, the leptin peptide (US provisional application No 60/155,506), or other compounds.
  • Assays include that described previously in Gavrilova et al. ((2000) Diabetes Nov;49(l 1): 1910-6; (2000) Nature Feb 24;403(6772):850) using A-ZTP/F-1 mice, except that GMG-1 polypeptides would be administered using the methods previously described in Example 3 (or Examples 6-8).
  • mice The glucose and insulin levels of the mice would be tested, and the food intake and liver weight monitored, as well as other factors, such as leptin, FFA, and TG levels, typically measured in our experiments (see Example 3, above, or Examples 6-8).
  • EXAMPLE 6 Effect of GMG-1 Polypeptides on Plasma Free Fatty Acid in C57 BL/6 Mice
  • mice used in this experiment are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken. All blood samples are taken from the tail using EDTA coated capillary tubes (50 ⁇ L each time point).
  • a GMG-1 polypeptide is injected i.p. in 100 ⁇ L saline.
  • the same dose 25 ⁇ g/mL in lOO ⁇ L
  • Confrol animals are injected with saline (3xl00 ⁇ L). Untreated and treated animals are handled in an alternating mode.
  • Plasma samples are taken in hourly intervals, and are immediately put on ice. Plasma is prepared by cenfrifugation following each time point. Plasma is kept at -20°C and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako). Due to the limited amount of plasma available, glucose is determined in duplicate using pooled samples. For each time point, equal volumes of plasma from all 8 animals per freatment group are pooled.
  • FFA free fatty acids
  • TG triglycerides
  • glucose is determined in duplicate using pooled samples. For each time point, equal volumes of plasma from all 8 animals per freatment group are pooled.
  • EXAMPLE 7 Effect of GMG-1 Polypeptides on Plasma Leptin and Insulin in C57 BL/6 Mice
  • GMG-1 polypeptides The effect of GMG-1 polypeptides on plasma leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice is tested.
  • the experimental procedure is the same as that described in Example 6, except that blood is drawn only at 0, 2 and 4 hours to allow for greater blood samples needed for the determination of leptin and insulin by RIA.
  • mice are fasted for 2 hours prior to the experiment after which a baseline blood sample is taken. All blood samples are taken from the tail using EDTA coated capillary tubes (100 ⁇ L each time point).
  • 25 ⁇ g of a GMG-1 polypeptide is injected i.p. in 100 ⁇ L saline.
  • the same dose 25 ⁇ g in lOO ⁇ L
  • Confrol animals are injected with saline (3xl00 ⁇ L). Untreated and freated animals are handled in an alternating mode.
  • Plasma samples are immediately put on ice and plasma is prepared by centrifugation following each time point. Plasma is kept at -20°C and free fatty acids (FFA) are determined within 24 hours using a standard test kit (Wako). Leptin and Insulin are determined by RIA (ML-82R and SRI-13R, LINGO Research, Inc., St. Charles, MO) following the manufacturer's protocol. However, only 20 ⁇ L plasma is used. Each determination is done in duplicate. Due to the limited amount of plasma available, leptin and insulin are determined in 4 pools of 2 animals each in both treatment groups.
  • FFA free fatty acids
  • EXAMPLE 8 Effect of GMG-1 Polypeptides on Plasma FFA. TG and Glucose in C57 BL/6 Mice The effect of GMG-1 polypeptides on plasma FFA, TG, glucose, leptin and insulin levels during postprandial lipemia (PPL) in normal C57BL6/J mice has been described. Weight loss resulting from GMG-1 polypeptides (2.5 ⁇ g/day) given to normal C57BL6/J mice on a high fat diet has also been shown (Example 3).
  • Plasma samples are immediately put on ice. Plasma is prepared by centrifugation following each time point. Plasma is kept at -20 °C and free fatty acids (FFA), triglycerides (TG) and glucose are determined within 24 hours using standard test kits (Sigma and Wako).
  • FFA free fatty acids
  • TG triglycerides
  • glucose are determined within 24 hours using standard test kits (Sigma and Wako).
  • mice plasma free fatty acids increase after intragasfric administration of a high fat/sucrose test meal. These free fatty acids are mostly produced by the activity of lipolytic enzymes i.e. lipoprotein lipase (LPL) and hepatic lipase (HL). In this species, these enzymes are found in significant amounts both bound to endothelium and freely circulating in plasma.
  • lipolytic enzymes i.e. lipoprotein lipase (LPL) and hepatic lipase (HL).
  • LPL lipoprotein lipase
  • HL hepatic lipase
  • Another source of plasma free fatty acids is hormone sensitive lipase (HSL) that releases free fatty acids from adipose tissue after ⁇ -adrenergic stimulation.
  • HSL hormone sensitive lipase
  • mice are injected with epinephrine.
  • mice Two groups of mice are given epinephrine (5 ⁇ g) by intraperitoneal injection.
  • a freated group is injected with a GMG-1 polypeptide (25 ⁇ g) one hour before and again together with epinephrine, while confrol animals receive saline.
  • Plasma is isolated and free fatty acids and glucose are measured as described above (Example 8).
  • Muscles are rinsed for 30 min in incubation media with oxygenation. The muscles are then transfe ⁇ ed to fresh media (1.5 mL) and incubated at 30°C in the presence of I ⁇ Ci/mL [1- 14 C] oleic acid (American Radiolabeled Chemicals). The incubation vials containing this media are sealed with a mbber septum from which a center well carrying a piece of Whatman paper (1.5 cm x 11.5 cm) is suspended.
  • the muscle is removed from the medium, and an aliquot of 0.5 mL medium is also removed.
  • the vials are closed again and 1 mL of 35% perchloric acid is injected with a syringe into the media by piercing through the mbber septum.
  • the C0 2 released from the acidified media is collected by the Solvable in the center well.
  • the Whatman paper is removed from the center well and placed in scintillation vials containing 15 mL of scintillation fluid (HionicFlour, Packard Instruments, Meriden, CT). The amount of 14 C radioactivity is quantitated by liquid scintillation counting.
  • the hindlimb muscle and liver triglyceride content is measured after the GMG-1 polypeptide freatment of mice.
  • Hind limb muscles as well as liver samples are removed from freated and untreated animals and the triglyceride and free fatty acid concentration is determined following a standard lipid extraction method (Shimabukuro,
  • mice Two groups of mice are intravenously (tail vein) injected with 30 ⁇ L bolus of Intralipid-20% (Clintec) to generate a sudden rise in plasma FFAs, thus by-passing intestinal abso ⁇ tion.
  • Intralipid is an intravenous fat emulsion used in nutritional therapy.
  • a freated group (GMG-1 polypeptide- freated) is injected with a GMG-1 polypeptide (25 ⁇ g) at 30 and 60 minutes before Infralipid is given, while control animals (D confrol) received saline. Plasma is isolated and FFAs are measured as described previously. The effect of GMG-1 polypeptides on the decay in plasma FFAs following the peak induced by Infralipid injection is then monitored.
  • L6 Muscle cells are obtained from the European Culture Collection (Porton Down) and are used at passages 7-11. Cells are maintained in standard tissue culture medium DMEM, and glucose uptake is assessed using [.sup.3 H]-2-deoxyglucose (2DG) with or without GMG-1 polypeptides in the presence or absence of insulin (10.sup.-8 M) as has been previously described (Walker, P.S. et al. (1990) Glucose fransport activity in L6 muscle cells is regulated by the coordinate control of subcellular glucose transporter distribution, biosynthesis, and mRNA transcription. JBC 265(3):1516-1523; and Rilp, A. et al.
  • EXAMPLE 14 In Vivo Tests for Metabolic-related Activity in Rodent Diabetes Models As metabolic profiles differ among various animal models of obesity and diabetes, analysis of multiple models is undertaken to separate the effects GMG-1 polypeptides on hyperglycemia, hyperinsulinemia, hyperlipidemia and obesity. Mutation in colonies of laboratory animals and different sensitivities to dietary regimens have made the development of animal models with noninsulin dependent diabetes associated with obesity and insulin resistance possible.
  • the fa/fa mutation may be the rat equivalent of the murine db mutation (Friedman et al., Cell 69:217-220, 1992; Truett et al., Proc. Natl. Acad. Sci. USA 88:7806, 1991).
  • Tubby (tub/tub) mice are characterized by obesity, moderate insulin resistance and hyperinsulinemia without significant hyperglycemia (Coleman et al., J. Heredity 81:424, 1990).
  • leptin was reported to reverse insulin resistance and diabetes mellitus in mice with congenital lipodysfrophy (Shimomura et al. Nature 401 : 73-76 (1999).
  • Leptin is found to be less effective in a different lipodysfrophic mouse model of lipoafrophic diabetes (Grajova et al Nature 403: 850 (2000); hereby inco ⁇ orated herein in its entirety including any drawings, figures, or tables).
  • the streptozotocin (STZ) model for chemically-induced diabetes is tested to examine the effects of hyperglycemia in the absence of obesity.
  • STZ-freated animals are deficient in insulin and severely hyperglycemic (Coleman, Diabetes 31:1, 1982; E. Shafrir, in Diabetes Mellitus; H. Rifkin and D. Porte, Jr. Eds. (Elsevier Science Publishing Co., Inc., New York, ed. 4, 1990), pp.
  • the monosodium glutamate (MSG) model for chemically-induced obesity (Olney, Science 164:719, 1969; Cameron et al., Cli. Exp. Pharmacol. Physiol. 5:41, 1978), in which obesity is less severe than in the genetic models and develops without hype ⁇ hagia, hyperinsulinemia and insulin resistance, is also examined.
  • a non-chemical, non-genetic model for induction of obesity includes feeding rodents a high fat/high carbohydrate (cafeteria diet) diet ad libitum.
  • the instant invention encompasses the use of GMG-1 polypeptides for reducing the insulin resistance and hyperglycemia in any or all of the above rodent diabetes models or in humans with
  • the GSSP4 polypeptides may, if desired, be associated with other compatible pharmacologically-active antidiabetic agents such as insulin, leptin (US provisional application No 60/155,506), or troglitazone , either alone or in combination.
  • Assays include that described previously in Gavrilova et al. ((2000) Diabetes Nov;49(ll):1910-6; (2000) Nature Feb 24;403(6772):850) using A-ZTP/F-1 mice, except that GSSP4 polypeptides are administered infraperotineally, subcutaneously, intramuscularly or intravenously.
  • mice In Vivo Assay for Anti-hvperglvcemic Activity of GMG-1 polypeptides
  • db/db Male, 7-9 weeks old
  • mice/cage 7-9 mice/cage
  • Purina rodent chow and water ad libitum Prior to treatment, blood is collected from the tail vein of each animal and blood glucose concenfrations are determined using One Touch BasicGlucose Monitor System (Lifescan).
  • mice that have plasma glucose levels between 250 to 500 mg/dl are used.
  • Each freatment group consists of seven mice that are distributed so that the mean glucose levels are equivalent in each group at the start of the study, db/db mice are dosed by micro-osmotic pumps, inserted using isoflurane anesthesia, to provide GMG-1 polypeptides, saline, and an i ⁇ elevant peptide to the mice subcutaneously (s.c).
  • Blood is sampled from the tail vein hourly for 4 hours and at 24, 30 h post-dosing and analyzed for blood glucose concenfrations. Food is withdrawn from 0-4 h post dosing and reinfroduced thereafter.
  • GMG-1 polypeptides or vehicle is administered through the jugular vein after complete recovery and for the following two days.
  • hyperinsulinemic-euglycemic clamps are performed. Rodents are placed in resfrainers and a bolus of 4 .mu Ci [3-.sup.3 H] glucose (NEN) is administered, followed by a continuous infusion of the tracer at a dose of 0.2 .mu.Ci/min (20 .mu.l/min).
  • 3 blood samples 0.3 ml each
  • An insulin infusion is then started (5 mU/kg/min), and 100 .mu.l blood samples are taken every 10 min. to monitor plasma glucose.
  • a 30% glucose solution is infused using a second pump based on the plasma glucose levels in order to reach and maintain euglycemia.
  • Glucose specific activity is determined in deproteinized plasma and the calculations of Rd and hepatic glucose output (HGO) are made, as described (Lang et al.,
  • Plasma insulin levels at basal period and after 5 and 25 mU/kg/min. infusions are then determined and compared between GMG-1 freated and vehicle treated rodents.
  • Insulin regulation of glucose homeostasis has two major components; stimulation of peripheral glucose uptake and suppression of hepatic glucose output. Using tracer studies in the glucose clamps, it is possible to determine which portion of the insulin response is affected by the
  • GMG-1 polypeptides are GMG-1 polypeptides.
  • Dipeptidyl peptidase cleavage of GMG-1 (47-285, 127-285 or 134-185 of SEQ ID NO: 2, or 47-217 of SEQ ID NO: 4) polypeptide fragment is determined by ELISA using a monoclonal antibody that specifically binds to intact GMG-1 polypeptide fragment but not to said GMG-1 polypeptide fragment from which the N-terminal dipeptide EP has been removed.
  • said antibody binds to GMG-1 (47-285, 127-285 or 134-185 of SEQ ID No: 2) but not to GMG-1 (49- 285, 129-285 or 136-185 of SEQ ID NO: 2) or said antibody binds to GMG-1 (47-217 of SEQ ED NO: 4) but not to GMG-1 (49-217 of SEQ ID NO: 4).
  • Dipetidyl peptidase is selected from but not restricted to human plasma comprised of dipeptidyl peptidase, soluble human CD26, or soluble human Attractin. Briefly, GMG-1 (5 ⁇ l, 100 fmol) is added to plasma samples (95 ⁇ l) and incubated for 1 h at
  • GMG-1 polypeptide fragment As a reference, GMG-1 polypeptide fragment (5 ⁇ l, 100 fmol) is added to heat-inactivated plasma (95 ⁇ l), which contains 0.01 mmol/1 valine-py ⁇ olidide and 500 RIE/ml aprotinin.
  • the extent of dipeptidyl peptidase cleavage of GMG-1 polypeptide fragment is calculated relative to the reference and expressed as a percentage.
  • EXAMPLE 16 Effect of gACRP30 on Maintenance of Weight Loss in Mice
  • mice are put on a reduced calorie diet to promote weight loss.
  • the reduced calorie diet is continued until the mice lose 10% of their intitial weight.
  • a second group of mice are continued on the weight reduced diet until the mice lose 20%> of their intitial weight.
  • the mice are then surgically implanted with an osmotic pump (Alzet, Newark, DE) delivering either 2.5 ⁇ g/day of gACRP30, 5 ⁇ g/day of ACRP30, or physiological saline.
  • the mice are returned to a normal diet and their body weights are recorded over a 10-day period. After 10 days, the result that mice that have been treated with gACRP30 have a lower weight than mice that were freated with saline will be taken to provide evidence that freatment with gACRP30 promotes the maintenance of weight loss.
  • Vector construction Polynucleotides encoding polypeptides selected from amino acids 16-285, 17- 285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-285, 71-285, 72-285, 73-2
  • Polynucleotides encoding polypeptides selected from amino acids 1-285 of SEQ ID NO:2 or amino acids 1-217 of SEQ ID NO:4 are cloned into Baculoviral expression vector FastBacHT.
  • Polynucleotides encoding polypeptides comprising amino acids 1-285 of SEQ ID NO:2 or amino acids 1-217 of SEQ ID NO:4 are cloned into mammalian expression vector pcDNA4HisMax.
  • Polynucleotides encoding polypeptides comprising amino acids 1-285 of SEQ ID NO:2 or amino acids 1-217 of SEQ ID NO:4 are cloned into mammalian expression vector pcDNA3.1Hygro.
  • polynucleotides encoding for polypeptides comprising amino acids 1-244, 16- 285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285, 61-285, 62-285, 63-285, 64-285, 65-285, 66-285, 67-285, 68-285, 69-285, 70-285, 71-285, 7
  • polynucleotides encoding a heterologous polypeptide comprised of human zinc-alpha 2-glycoprotein signal peptide fused N-terminally to gGMG-1 polypeptide fragment of the invention wherein said gGMG-1 polypeptide fragment is selected from amino acids 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26- 285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-
  • polynucleotides encoding for polypeptides selected from amino acids 1-285 are also known.
  • polypeptides selected from amino acids 1-285 are also known.
  • polynucleotides encoding a heterologous polypeptide comprised of human zinc-alpha 2-glycoprotein signal peptide fused N-terminally to gGMG-1 polypeptide fragment of the invention wherein said gGMG-1 polypeptide fragment is selected from 16-285, 17-285, 18-285, 19-285, 20-285, 21-285, 22-285, 23-285, 24-285, 25-285, 26-285, 27-285, 28-285, 29-285, 30-285, 31-285, 32-285, 33-285, 34-285, 35-285, 36-285, 37-285, 38-285, 39-285, 40-285, 41-285, 42-285, 43-285, 44-285, 45-285, 46-285, 47-285, 48-285, 49-285, 50-285, 51-285, 52-285, 53-285, 54-285, 55-285, 56-285, 57-285, 58-285, 59-285, 60-285,
  • EXAMPLE 18 Infant formula supplementation.
  • the following example describes a method of administering gGMG-1 polypeptides to newborns as supplemental nutritional support and further provides a method of promoting growth of an infant by administering gGMG-1 -fortified human breast milk, or gGMG-1 -fortified breast milk substitute formulation from a nonhuman source.
  • Dehydrated or lyophilized gGMG-1 polypeptide powder is directly added to pumped human breast milk (freshly pumped or prewarmed after storage) or any prewarmed breast milk substitute formulation from a nonhuman source, in a range of 5-1000 ng/ml, preferably 20-800 ng/ml, more preferably 65-650 ng/ml.
  • Supplementation with polypeptides of the invention is provided to infants, particularly preterm infants, in bottle feedings of human breast milk or breast milk substitute at every feeding throughout the day, and is continued to be provided from birth to 6 months of age.
  • Preterm infants may be of low birth weight or very low birth weight.
  • a primary objective of the study is to demonstrate that a polypeptide of the invention added to human milk (HM) or human milk substitute (HMS) supports acceptable growth in preterm infants.
  • a second objective is to evaluate the semm biochemistries (ie, protein status, calcium, alkaline phosphatase), tolerance, clinical problems, and morbidity of premature infants consuming the nutritional module.
  • Another secondary objective is to compare the supplemental composition of the instant invention to a commercial fortifier powder that has been in use for a number of years to promote growth in preterm infants.
  • An intent-to-treat, prospective, randomized, double-blinded multicenter study is conducted to evaluate preterm infants receiving preterm milk supplemented with either a commercially available powdered human milk fortifier (EnfamiLRTM. Human Milk Fortifier, control) or the supplemental polypeptide (test) powder of the cu ⁇ ent invention
  • Study Day 1 is when fortification of the test powder begins and the subject reaches an intake of at least 100 mL/kg/day.
  • Anthropometric indices, semm biochemistries, intake, tolerance, and morbidity data are assessed.
  • Each infant is studied until hospital discharge; only anthropometric variables (weight, length, and head circumference) are collected after Study Day 29.
  • Premature infants are recruited from neonatal intensive care units that had agreed to collaborate with study investigators. Single, twin, or triplet infants bom around 33 weeks gestational age, with appropriate weight for gestational age, and weighing around 1600 g are eligible to participate.
  • One- hundred and forty-four infants are randomized to either control or experimental; 70 preterm infants are randomized to the control group and 74 preterm infants are randomized to the experimental group.
  • the randomization is proportional for birth weight and gender.
  • the independent variables (treatments) are the confrol fortifier powder and the experimental test powder which are added to HM or HMS. Both fortifiers (test and confrol) are provided in small packets in powdered form and are added to 25 mL HM or HMS.
  • the primary outcome variable is weight gain (g/kg/day) from study day 1 to study day 29 or discharge, whichever comes first.
  • Secondary outcome variables are length gain (mm/day) and semm biochemistries to evaluate protein status, electrolyte status, mineral homeostasis, and vitamin A and E status. Semm biochemistries also include unscheduled laboratory results to be recorded in the medical chart. Tertiary variables include head circumference gain (mm/day), clinical history, intake, tolerance, clinical problems/morbidity, respiratory status, antibiotic use, and the number of transfusions. Mean total energy intakes during the study period is not different between the groups, around 118 kcal/kg/day.
  • EXAMPLE 19 Assessment of homotrimer formation by gGMG-1 polypeptide fragment.
  • gGMG-1 polypeptide fragment homofrimer formation by gGMG-1 polypeptide fragment is assessed using sedimentation equilibrium in analytical centrifuges, a method that determines molecular weight accurately and independently of other physical factors such as shape.
  • Candidate gGMG-1 polypeptide fragment homofrimer is purified, for example using a protocol comprising a method of gel filtration such as 16/60 superdex 200 gel filtration column (Amersham). Said purified candidate gGMG-1 polypeptide fragment homofrimer protein concenfration is made 3 ⁇ M in 5.7 mM phosphate (pH 7.5), 137 mM NaCl, 2.7 mM RC1.
  • PRC ⁇ was assessed by Western blot analysis using an affinity-purified PKC antibody that recognizes a conserved hydrophobic C-terminal FXZF(S/I)(F/Y) motif only when the serine/threonine residues are phosphorylated (Cell Signaling Technology). Total amounts of protein in the individual lanes are normalized by Western blotting using a monoclonal antibody against ⁇ - tubulin (Sigma).
  • EXAMPLE 21 Effect of GMG-1 Polypeptides on NF- ⁇ B Activation.
  • Activation of NF- ⁇ B by GMG-1 Polypeptide Luciferase activity is measured in transfected cells following overnight incubation with 200 ng/ml LPS (E. coli serotype 055 :B5, Sigma) or 5 ⁇ g/ml GMG-1 polypeptide before and after proteinase K and heat freatment.
  • Cells are fransfected with an E-selectin promoter-luciferase construct [Schindler U et al.
  • Cells are freated with 200 ng/ml LPS or 5 ⁇ g/ml GMG-1 polypeptide in normal growth media. After 30 and 120 min of incubation, cells are washed with PBS containing Ca 2+ and Mg 2+ and lysed as described herein. The amount of phosphorylated and total I ⁇ B- ⁇ in lysates is assessed by Western blot analysis using affinity purified Ser32 phospho-specific antibody and a different phosphorylation state-independent I ⁇ B- ⁇ antibody (Cell Signaling Technology).
  • EXAMPLE 22 Effect of iAFLP Gene Expression Profiling Analysis of Poly A+ and Total RNA from Different Tissue Sources
  • IAFLP Amplified Fragment Length Polymo ⁇ hism
  • RNA is DNase freated for 30 minutes at 37C.
  • 20 ug of total RNA or 2 ug of Poly A+ RNA is converted to dscDNA using the cDNA Synthesis System supplied by Roche
  • the purified and quantified dscDNA is cleaved using the Mbol restriction enzyme kit supplied by New England Biolabs as per the manufacture's instructions.
  • One-third of the Mbol cleaved dscDNA is ligated to kinated adaptor cassette primers using the T4 DNA Ligation kit supplied by Roche Applied Science as per the manufacture's instructions.
  • the ligated dscDNA is diluted in glycogen and DI water to a final concenfration of lng/ul.
  • One ng is added to a final PCR master mix volume of lOul containing 0.2mM dNTPs, lmM PCR Buffer, 2mM MgC12, 2uM Vic fluorescent labeled T7 primer, 2uM of a gene-specific reverse primer, 8% glycerol, and lUnit Amplitaq Gold DNA Polymerase.
  • the template is incubated for 10 min at 95C, denatured for 30 sec.at 95C, annealed for 1 min at 60C, extended for 30 sec. at 72C for 35 cycles and extended 7 min at 72C for 1 cycle.
  • PCR reaction is performed using the Applied Biosystem's 9700 GeneAmp thermalcycler.
  • One ul of the PCR reaction is diluted 1:100.
  • 1 ul of the diluted PCR product is combined with 0.1 ul of the Liz 500 size standard (Applied Biosystems) and 8.9 ul of HiDi Formamide (Applied Biosystems).
  • the mixture is denatured for 5 min. at 95C.
  • 1 ul of denatured mixture is loaded into a 3700 DNA Analyzer (Applied Biosystems) and separated by size. Analysis of the differently sized fragments is performed by the Genescan software package supplied by Applied Biosystems as per the manufacture's instructions.

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Abstract

La présente invention a trait au domaine de la recherche contre l'obésité, problème de santé publique grave et répandu. Un composé, homotrimère d'un fragment de polypeptide GMG-1 comprenant un domaine globulaire et tout ou partie d'un domaine GMG-1 de type collagène, a été identifié ; ce dernier permet de réduire la masse corporelle, de maintenir la perte de poids, et de traiter des maladies et des troubles liés à l'obésité. Ces maladies et troubles liés à l'obésité comprennent les hyperlipidémies, l'athérosclérose, le diabète et l'hypertension.
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WO2003102027A1 (fr) * 2002-05-31 2003-12-11 Serono Genetics Institute S.A. Tete globulaire de obg3 etendue et homotrimere et utilisations associees
WO2007014798A2 (fr) * 2005-07-29 2007-02-08 Laboratoires Serono S.A. Utilisation d'acrp30 pour le traitement et/ou la prevention de la thrombose et du cancer
WO2007102736A2 (fr) * 2006-03-08 2007-09-13 Universitair Medisch Centrum Utrecht Procédé d'interférence dans l'activation d'une cellule immunitaire par commande de l'interaction entre lair et collagène
US7638638B2 (en) 2003-05-14 2009-12-29 Takeda San Diego, Inc. Dipeptidyl peptidase inhibitors
US7960384B2 (en) 2006-03-28 2011-06-14 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US8084605B2 (en) 2006-11-29 2011-12-27 Kelly Ron C Polymorphs of succinate salt of 2-[6-(3-amino-piperidin-1-yl)-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethy]-4-fluor-benzonitrile and methods of use therefor
US8093236B2 (en) 2007-03-13 2012-01-10 Takeda Pharmaceuticals Company Limited Weekly administration of dipeptidyl peptidase inhibitors
US8222411B2 (en) 2005-09-16 2012-07-17 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US8324383B2 (en) 2006-09-13 2012-12-04 Takeda Pharmaceutical Company Limited Methods of making polymorphs of benzoate salt of 2-[[6-[(3R)-3-amino-1-piperidinyl]-3,4-dihydro-3-methyl-2,4-dioxo-1(2H)-pyrimidinyl]methyl]-benzonitrile
WO2014016362A1 (fr) 2012-07-24 2014-01-30 Sanofi Pasteur Compositions de vaccin pour prévenir une infection provoquée par le virus de la dengue
US8906901B2 (en) 2005-09-14 2014-12-09 Takeda Pharmaceutical Company Limited Administration of dipeptidyl peptidase inhibitors

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WO2004087053A2 (fr) 2003-03-25 2004-10-14 Syrrx, Inc. Inhibiteurs de dipeptidyle peptidase
DE602004010206T2 (de) 2003-08-13 2008-10-09 Takeda Pharmaceutical Co. Ltd. Dipeptidyl Peptidase Inhibitoren.

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003102027A1 (fr) * 2002-05-31 2003-12-11 Serono Genetics Institute S.A. Tete globulaire de obg3 etendue et homotrimere et utilisations associees
US7459433B2 (en) 2002-05-31 2008-12-02 Serono Genetics Institute, S.A. Homotrimeric extended OBG3 globular head and uses thereof
US7638638B2 (en) 2003-05-14 2009-12-29 Takeda San Diego, Inc. Dipeptidyl peptidase inhibitors
WO2007014798A2 (fr) * 2005-07-29 2007-02-08 Laboratoires Serono S.A. Utilisation d'acrp30 pour le traitement et/ou la prevention de la thrombose et du cancer
WO2007014798A3 (fr) * 2005-07-29 2007-08-23 Serono Lab Utilisation d'acrp30 pour le traitement et/ou la prevention de la thrombose et du cancer
US8906901B2 (en) 2005-09-14 2014-12-09 Takeda Pharmaceutical Company Limited Administration of dipeptidyl peptidase inhibitors
US8222411B2 (en) 2005-09-16 2012-07-17 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
WO2007102736A2 (fr) * 2006-03-08 2007-09-13 Universitair Medisch Centrum Utrecht Procédé d'interférence dans l'activation d'une cellule immunitaire par commande de l'interaction entre lair et collagène
WO2007102736A3 (fr) * 2006-03-08 2008-03-13 Univ Medisch Centrum Utrecht Procédé d'interférence dans l'activation d'une cellule immunitaire par commande de l'interaction entre lair et collagène
US7960384B2 (en) 2006-03-28 2011-06-14 Takeda Pharmaceutical Company Limited Dipeptidyl peptidase inhibitors
US8324383B2 (en) 2006-09-13 2012-12-04 Takeda Pharmaceutical Company Limited Methods of making polymorphs of benzoate salt of 2-[[6-[(3R)-3-amino-1-piperidinyl]-3,4-dihydro-3-methyl-2,4-dioxo-1(2H)-pyrimidinyl]methyl]-benzonitrile
US8084605B2 (en) 2006-11-29 2011-12-27 Kelly Ron C Polymorphs of succinate salt of 2-[6-(3-amino-piperidin-1-yl)-3-methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-ylmethy]-4-fluor-benzonitrile and methods of use therefor
US8093236B2 (en) 2007-03-13 2012-01-10 Takeda Pharmaceuticals Company Limited Weekly administration of dipeptidyl peptidase inhibitors
WO2014016362A1 (fr) 2012-07-24 2014-01-30 Sanofi Pasteur Compositions de vaccin pour prévenir une infection provoquée par le virus de la dengue

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