WO1999023115A1 - Analogues de la proteine de l'obesite glycosylee - Google Patents

Analogues de la proteine de l'obesite glycosylee Download PDF

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Publication number
WO1999023115A1
WO1999023115A1 PCT/US1998/023323 US9823323W WO9923115A1 WO 1999023115 A1 WO1999023115 A1 WO 1999023115A1 US 9823323 W US9823323 W US 9823323W WO 9923115 A1 WO9923115 A1 WO 9923115A1
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replaced
asp
asn
glu
lys
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PCT/US1998/023323
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English (en)
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John Michael Beals
John Edward Hale
Steven Duane Hatch
Joseph Vincent Junior Rinella
Brigitte Elisabeth Schoner
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Eli Lilly And Company
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Priority to AU12085/99A priority Critical patent/AU1208599A/en
Publication of WO1999023115A1 publication Critical patent/WO1999023115A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is in the field of human medicine, particularly in the treatment of obesity and disorders associated with obesity. More specifically, the present invention relates to glycosylated obesity proteins. 2. Background Information.
  • Obesity, and especially upper body obesity, is a common and very serious public health problem in the United States and throughout the world, and is expected to worsen as the population ages. Currently, about 33% of Americans are overweight enough to be unhealthy ( i . e . , body weight greater than 26 percent above standard weight guidelines) . For comparison, about 25% of Americans were overweight by these criteria in 1980 [United States National Center for Health Statistics] . The proportion of obese adults among the well-fed populations of the world is expected to rise to more than 50% within 20 years .
  • the obesity protein regulates adiposity [Pelleymounter et al . , Science 269:540-543 (1995); Zhang et al . Nature 372:425-32 (1994); Murakami et al . , Biochemical and Biophysical Research Communications 209 (3) : 944-52 (1995)] . Unfortunately, the obesity protein disclosed by Zhang et al .
  • the objective of the present invention is to increase the plasma half-life of obesity protein in the circulation and to improve the chemical and physical stability of obesity protein formulations in order to provide a more suitable obesity protein for the effective pharmaceutical treatment of the severe problem of obesity.
  • glycoproteins For a glycosylated protein, specific oligosaccharide sequences govern the clearance of glycoproteins from the circulation by targeting it to receptors on particular cell-types for removal from the circulation and thereby affecting the protein's half-life in the circulation [Ashwell, G., et al . , Adv. Enzymol . 41:99- 128 (1974); Prieels, J.-P., et al . Proc . Natl . Acad. Sci . USA 75:2215-2219 (1978); Steer, C. J., et al . , J. Biol .
  • N represents asparagine, the amino acid that is actually linked to a glycosyl group
  • X represents any amino acid except aspartic acid, tryptophan, or proline
  • T represents threonine
  • S represents serine.
  • Glycosylation sites in Mitchell, et al . may be introduced at one or more positions. In selecting positions for glycosylation, Mitchell, et al . endeavored to retain IL-2 biological activity, and to conserve the structure of the region in which the modification was made. IL-2 analogs having glycosylation sites introduced into them were glycosylated to varying degrees when expressed in cultured HeLa cells. Mitchell, et al . provide no data on physical stability of formulations of glycosylated IL-2, nor do they provide any in vivo data on the persistence of glycosylated IL-2 in the circulation.
  • introducing glycosylation sites include tissue plasminogen activator (tPA) [Bennett, et al . , U.S. Patent No. 5,612,029, issued 18 Mar 1997; Bell, L. D., et al . , U.S. Patent No. 5,244,676, issued 14 Sep 1993; Larsen, G. R. , et al . , U.S. Patent No. 5,258,298, issued 2 Nov 1993], lysozyme [Hummel, et al . , Eur. J. Biochem. 245:428-433 (1997); Horst, M., et al . , J. Biol . Chem.
  • tPA tissue plasminogen activator
  • yeast invertase [Schulke, N. , et al . , J. Biol . Chem. 263:8827-8831 (1988); Kern, G. , et al . ,
  • Replacing or deleting glutamine residues and asparagine residues that are not at glycosylation sites may improve the chemical stability of the obesity protein. Additionally, glycosylating at asparagine residues may improve the chemical stability of the obesity protein by eliminating deamidation sites. Finally, glutamine residues can be conservatively substituted with asparagine residues targeted for glycosylation, thereby eliminating potential deamidation sites.
  • Obesity protein is not glycosylated when expressed in vivo in mammalian cells capable of glycosylating at consensus N-linked glycosylation sites. Thus, the effect of glycosylating obesity protein on its persistence in the circulation and on its physical stability was not previously known or described.
  • a significant aspect of the present invention are glycosylated obesity proteins and glycosylated obesity protein analogs that have longer half-life in the circulation or increased physical stability, or both.
  • This invention provides obesity protein analogs having substantially the same amino acid sequence as human obesity protein, but differing from human obesity protein in having one or more consensus N-linked glycosylation sites, or a pharmaceutically acceptable salt thereof .
  • the obesity protein analogs optionally have amino acid replacements, deletions, or both, to improve chemical, or physical stability, or both.
  • the obesity protein analogs are useful themselves for treating obesity, and are also useful as intermediates in preparing glycosylated proteins of the present invention.
  • the invention further provides glycosylated proteins, selected from the group consisting of glycosylated obesity protein and glycosylated obesity protein analogs, or a pharmaceutically acceptable salt thereof.
  • glycosylated proteins of the present invention are useful for treating obesity.
  • the glycosylated obesity proteins have increased plasma half-life and improved physical stability compared with non-glycosylated obesity proteins.
  • the invention further provides a DNA polynucleotide compound comprising a DNA nucleotide sequence encoding the amino acid sequence of the obesity protein analog of the present invention.
  • the DNA polynucleotide compound is useful as an intermediate in preparing the obesity protein analogs of the present invention.
  • the invention further provides a formulation of the glycosylated proteins of the present invention, comprising, a glycosylated protein of the present invention and a preservative .
  • Also part of the invention is a method of treating obesity, comprising administering an effective dose of a glycosylated protein of the present invention to a patient in need thereof .
  • a method for improving the biological or physical properties of an obesity protein for pharmaceutical use comprising, creating a glycosylation site in the protein, and glycosylating the obesity protein at the glycosylation site.
  • Fig. 1 depicts typical N-linked carbohydrates of glycoproteins , where SA is sialic acid, Gal is galactose, Man is mannose, GlcNAc is N-acetyl glucosamine, and Asn is an asparagine residue in a glycosylated protein.
  • Fig. 2 depicts N-terminal glycosylation linkages, and a scheme for glycosylating at the N-terminus.
  • administration means the introduction of a substance into the body of a patient.
  • Administration of the compounds and formulations of the present invention may be via any route known to be effective by the physician of ordinary skill.
  • Parenteral administration is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump.
  • Routes of administration include, without limitation, intravenous, intramuscular, subcutaneous, intraperitoneal, oral, nasal, pulmonary, vaginal, occular, buccal, transdermal routes, as well as, delivery by gene therapy wherein the protein is produced within the body of the patient.
  • base pair which may be abbreviated as “bp” refers to a pair of DNA nucleotide bases that are hydrogen-bonded to each other in a double-stranded DNA polynucleotide.
  • the abbreviations A, C, G, and T correspond to the 5 ' -monophosphate forms of the nucleotides
  • glycosylate means to form a bond between an obesity protein or an obesity protein analog and a glycosyl moiety to form a "glycosylated obesity protein” or a “glycosylated obesity protein analog,” respectively.
  • a “glycosylated obesity protein” has a glycosyl moiety covalently bonded to a protein that has the amino acid sequence of human obesity protein.
  • a “glycosylated obesity protein analog” has a glycosyl moiety covalently bonded to a protein that has an amino acid sequence differing from the amino acid sequence of human obesity protein in having one or more amino acid modifications.
  • the glycosyl moiety may be covalently bonded to an obesity protein or to an obesity protein analog via certain nitrogen atoms in the protein. Bonding of the glycosyl group to a nitrogen atom [Jeong, S. Y., et al . , J.
  • Controlled Release 1:57-66 (1984)] may be via the N-terminal ⁇ -amino nitrogen or an ⁇ -amino group of a lysine residue. Additionally, the glycosyl moiety may be covalently attached to the amide nitrogen atom of an asparagine residue in an obesity protein analog if that asparagine is at a consensus N-linked glycosylation site that is introduced into the analog .
  • the glycosyl moiety may vary, from a monosaccharide, such as, without limitation, glucose or mannose, to complex oligosaccharide moieties, such as, without limitation, the complex, hybrid, and high-mannose type of oligosaccharides demonstrated in Figure 1.
  • oligosaccharide moieties such as, without limitation, the complex, hybrid, and high-mannose type of oligosaccharides demonstrated in Figure 1.
  • heterogeneity is observed within the population of glycoprotein molecules biosynthesized in vivo [Wright, A. et al . , Trends in Biotechnol . 15:26-32 (1997); Hummel, M., et al . , Eur. J. Biochem. 245:428-433 (1997); Horst, M. , et al . , J. Biol . Chem . , 268:19690-19696 (1993); Lis, H., et al .
  • Such heterogeneity may be influenced by the availability of the glycosylation site on the surface of the protein for transfer of oligosaccharide moieties, the genotype and phenotype of the host cell line, and also by environmental conditions during expression.
  • Carbohydrate structures in yeast-produced glycoproteins are summarized in Tanner, W., et al . , Biochim . Biophys . Acta 906:81-99 (1987), the entire disclosure of which is incorporated herein by reference.
  • the oligosaccharide moieties of mammalian plasma proteins are summarized in Baenziger, J. U., The Plasma Proteins : Structure, Function, and Genetic Control , Putnam, F. W.
  • glycosyl moiety only refers to naturally expressed or synthesized oligosaccharides. Therefore, the term does not include oligosaccharides not expressed or synthesized in vivo, such as, polyethylene glycol moieties or dextran moieties.
  • glycosylation site refers to the amino acid position or to the amino acid residue that is actually glycosylated.
  • a "consensus N-linked glycosylation site” has an amino acid sequence NXT or NXS, where N represents asparagine, X represents any amino acid except proline, T represents threonine, and S represents serine.
  • N represents asparagine
  • X represents any amino acid except proline
  • T represents threonine
  • S serine.
  • This type of glycosylation site is recognized by glycosyltransferase enzymes that are present in many eukaryotic cells. Glycosylation occurs at the asparagine residue, and glycosylation sites are described by the position of the asparagine residue.
  • Naturally-occurring human obesity protein does not contain a consensus N-linked glycosylation site.
  • a consensus N-linked glycosylation site may be created in several ways in an obesity protein analog. Replacement of a single amino acid two residues in the C- terminal direction from one of the four asparagine residues in human obesity protein with serine or threonine produces a consensus N-linked glycosylation site. Substituting asparagine for the amino acid two residues in the N-terminal direction from one of the seventeen serine residues or from one of the eleven threonine residues also creates a consensus N-linked glycosylation site.
  • a consensus N-linked glycosylation site may be created at any other position, except the last two at the C-terminus, by substituting an asparagine at that position, and by simultaneously replacing the amino acid two residues in the C-terminal direction with serine or threonine. If proline is at the +1 position from a desired glycosylation site, the proline must be replaced with any amino acid other than proline to create a consensus N-linked glycosylation site. At certain positions, deletion of a single amino acid will create a consensus N-linked glycosylation site. Finally, insertion of a single amino acid, either Asn, or Ser or Thr, and optionally replacement of the appropriate amino acid with Ser or Thr, or with Asn, respectively, will create a consensus N-linked glycosylation site.
  • isotonicity agent refers to an agent that is tolerated physiologically and imparts a suitable tonicity to the formulation to prevent the net flow of water across the cell membrane.
  • Compounds, such as glycerin, are commonly used for such purposes at known concentrations .
  • Other possible isotonicity agents include salts, e.g., NaCl, dextrose, mannitol, and lactose.
  • human obesity protein refers to the predominant allelic variant of obesity protein found in human populations. Human obesity protein that has the amino acid sequence of SEQ ID NO:l:
  • the term "obesity protein analog” refers to a protein that has an amino acid sequence differing from the amino acid sequence of human obesity protein in having one or more amino acid modifications.
  • Obesity protein analogs are known in the art, and include non-human obesity proteins, such as, the rat obesity protein [Murakami et al . , Biochemical and Biophysical Research Comm. 209:944-952 (1995)], porcine and bovine obesity proteins [Hansen, H. M., et al . , U.S. Application No. 08/445,305, (EPO 743 321)], and other obesity proteins [Basinski, M., et al . , U.S. Application No.
  • amino acid “modification” refers to a change in the amino acid sequence of human obesity protein, including: 1) replacing one or more amino acids with different amino acids; 2) deleting one or more amino acids; and 3) adding one or more amino acids.
  • hOb is an abbreviation for human obesity protein, whose amino acid sequence is given by SEQ ID NO:l.
  • the term “obesity protein analogs of the present invention” refers to a subset of obesity protein analogs, specifically, those having a consensus N-linked glycosylation site. Abbreviations for designating the obesity protein analogs of the present invention will be used herein.
  • Q4N-hOb represents an obesity protein analog having the sequence of human obesity protein, except at position 4, where asparagine (N) replaces glutamine (Q) .
  • n( ⁇ )n+l an abbreviation of the form "n( ⁇ )n+l” is used, wherein, “n” and “n+1” are the positions between which the insertion is made, and “ ( ⁇ ) " represents, in one-letter coding, the amino acid, or sequence of amino acids, inserted between positions "n” and “n+1.”
  • insertion of any amino acid between positions 28 and 29 is represented by "28(-P)29” and insertion of the dipeptide Asn-Leu between positions 30 and 31 is represented by "30(NL)31.”
  • one of the inserted amino acids is an asparagine in a consensus N-linked glycosylation site
  • the site is designated as "n.5.”
  • the insertion "35N36” creates a consensus glycosylation site designated as "35.5.”
  • buffer or “pharmaceutically-acceptable buffer” refers to a compound that is known to be safe for use in pharmaceutical formulations and that has the effect of controlling the pH of the formulation at the pH desired for the formulation.
  • Pharmaceutically-acceptable buffers for controlling pH at a moderately acidic pH to a moderately basic pH include such compounds as phosphate, acetate, citrate, arginine, TRIS, and histidine.
  • TRIS tritrigly acceptable salt thereof .
  • the free base and the hydrochloride form are two common forms of TRIS.
  • TRIS is also known in the art as trimethylol aminomethane, tromethamine, and tris (hydroxymethyl) aminomethane.
  • Other buffers that are pharmaceutically acceptable, and that are suitable for controlling pH at the desired level will be known to the chemist of ordinary skill .
  • a "plasmid” is an extrachromosomal self- replicating genetic element.
  • preservative refers to a compound added to a pharmaceutical formulation to act as an anti-microbial agent .
  • a parenteral formulation must meet guidelines for preservative effectiveness to be a commercially viable multi-use product.
  • preservatives known in the art as being effective and acceptable in parenteral formulations are benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercurie nitrate, thimerosal, benzoic acid, and various mixtures thereof. See, e.g., WALLHAUSER, K.
  • phenolic preservative includes the compounds phenol, m-cresol, o-cresol, p-cresol, chlorocresol, methylparaben, and mixtures thereof .
  • reading frame refers to the nucleotide sequence from which translation occurs when "read” in triplets by the translational apparatus of tRNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid. Because each triplet is distinct and of the same length, the coding sequence must be a multiple of three. A base pair insertion or deletion (termed a frameshift mutation) may result in two different proteins being coded for by the same DNA segment . To insure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i . e . , the "correct reading frame" must be maintained.
  • N-terminal-fusion proteins that are comprised of an obesity protein or an analog thereof, together with a leader peptide or protein at the N- terminus, the reading frame of the DNA sequence, starting from the end corresponding to the N-terminus of the fusion, must be maintained in the DNA sequence encoding the fusion peptide.
  • a "recombinant DNA cloning vector” is any autonomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
  • a "recombinant DNA expression vector” is any recombinant DNA cloning vector in which a promoter has been incorporated.
  • the term “replicon” refers to a DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
  • the term “soluble” means the relative absence of aggregated protein that is visually perceivable.
  • the degree of aggregation of formulations of proteins may be inferred by measuring the turbidity or the light scattering intensity of the formulation. The greater the turbidity of the formulation, the greater the extent of aggregation of the protein in the formulation. Turbidity is commonly determined by nephelometry, and measured in Nephalometric Turbidity Units (NTU) .
  • Transcription refers to the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence .
  • Translation refers to the process whereby the genetic information of messenger RNA is used to specify and direct the synthesis of a polypeptide chain.
  • treating describes the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a formulation of the present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating as used herein includes the administration of the protein for cosmetic purposes.
  • a cosmetic purpose seeks to control the weight of a mammal to improve bodily appearance.
  • a "vector” is a replicon used for the transformation of cells in gene manipulation bearing DNA polynucleotide sequences corresponding to appropriate protein molecules which, when combined with appropriate control sequences, confer specific properties on the host cell to be transformed. Plasmids, viruses, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. Vectors include Recombinant DNA cloning vectors and Recombinant DNA expression vectors .
  • the nucleotide and amino acid abbreviations used herein are those accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. ⁇ 1.822 (b) (2) (1993) , or are related to those abbreviations herein.
  • the one-letter code for amino acids will be familiar to the biochemist of ordinary skill. Unless otherwise indicated the amino acids are in the L configuration.
  • the present invention provides obesity protein analogs having consensus N-linked glycosylation sites.
  • a preferred group of obesity protein analogs of the present invention have such glycosylation sites created at particular positions in the sequence, and have one or more modifications selected from a first set of modifications, and optionally, one or more modifications selected from a second set of modifications.
  • the modifications in the first set are those that create a consensus N-linked glycosylation site in the protein at particular positions.
  • a preferred group of obesity protein analogs of the present invention are those having one or more modifications selected from the set below that create an N- linked glycosylation site or sites in the protein:
  • a preferred group of obesity protein analogs of the present invention consists of analogs having a consensus N-linked glycosylation site at one or more of the following positions: 4, 7, 8, 9, 10, 14, 17, 22, 23, 25, 27, 27.5, 28, 29, 29.5, 30, 30.5, 34. 35, 35.5, 40, 48, 48.5, 50, 55, 56, 62, 63, 64, 65, 68, 72, 75, 78, 79, 82, 85, 91, 93, 100, 101, 104, 105, 107, 108, 115, 118, 119, 125, 130, 134, 135, 139, and 141,
  • Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr
  • Gin at position 4 is replaced with Asn and Val at position 6 is replaced with Ser or Thr;
  • Gin at position 7 is replaced with Asn and Asp at position 9 is replaced with Ser or Thr; Asp at position 8 is replaced with Asn; Asp at position 8 is replaced with Asn and Thr at position 10 is replaced with Ser;
  • Asp at position 9 is replaced with Asn and Lys at position 11 is replaced with Ser or Thr; Thr at position 10 is replaced with Asn;
  • Thr at position 10 is replaced with Asn and Thr at position 12 is replaced with Ser; lie at position 14 is replaced with Asn; lie at position 14 is replaced with Asn and Thr at position 16 is replaced with Ser; lie at position 17 is replaced with Asn; lie at position 17 is replaced with Asn and Thr at position 19 is replaced with Ser;
  • Asp at position 23 is deleted or is replaced with Asn, and in either case, Ser at position 25 is replaced with Thr; lie at position 24 is deleted or is replaced with Ser or Thr;
  • Ser at position 25 is replaced with Asn and Thr at position 27 is replaced with Ser;
  • Gin at position 28 is replaced with Asn and Ser at position 29 is deleted; Gin at position 28 is replaced with Asn and Val at position 30 is deleted;
  • Gin at position 28 is replaced with Asn and any amino acid except Pro is inserted between position 28 and position 29; Gin at position 28 is replaced with Asn and Val at position 30 is replaced with Ser or Thr; Ser at position 29 is replaced with Asn;
  • Ser at position 29 is replaced with Asn and Ser at position 31 is replaced with Thr;
  • Val at position 30 is replaced with Asn and Ser at position 32 is replaced with Thr;
  • Gin at position 34 is replaced with Asn and Lys at position 35 is deleted;
  • Gin at position 34 is replaced with Asn and Val at position 36 is replaced with Ser or Thr; Lys at position 35 is replaced with Asn;
  • Lys at position 35 is replaced with Asn and Thr at position 37 is replaced with Ser;
  • Asp at position 40 is replaced with Asn and lie at position 42 is replaced with Ser or Thr; lie at position 48 is replaced with Asn; lie at position 48 is replaced with Asn and Thr at position 50 is replaced with Ser;
  • Thr at position 50 is replaced with Asn and Ser at position 52 is replaced with Thr;
  • Gin at position 56 is replaced with Asn and Leu at position 58 is replaced with Ser or Thr;
  • Gin at position 62 is replaced with Asn and lie at position 64 is replaced with Ser or Thr; Gin at position 63 is replaced with Asn and Leu at position 65 is replaced with Ser or Thr; lie at position 64 is replaced with Asn; lie at position 64 is replaced with Asn and Thr at position 66 is replaced with Ser;
  • Leu at position 65 is replaced with Asn; Leu at position 65 is replaced with Asn and Ser at position 67 is replaced with Thr;
  • Met at position 68 is replaced with Asn and Pro at position 69 is replaced with any amino acid except Pro;
  • Met at position 68 is replaced with Asn
  • Pro at position 69 is replaced with any amino acid except Pro
  • Ser at position 70 is replaced with Thr
  • lie at position 74 is replaced with Ser or Thr;
  • Gin at position 75 is replaced with Asn
  • Gin at position 75 is replaced with Asn and Ser at position 77 is replaced with Thr;
  • Asp at position 79 is replaced with Asn and Glu at position 81 is replaced with Ser or Thr;
  • Leu at position 80 is replaced with Ser or Thr;
  • Ala at position 91 is replaced with Asn
  • Ala at position 91 is replaced with Asn and Ser at position 93 is replaced with Thr; Ser at position 93 is replaced with Asn;
  • Ser at position 93 is replaced with Asn and Ser at position 95 is replaced with Thr;
  • Trp at position 100 is replaced with Asn
  • Trp at position 100 is replaced with Asn and Ser at position 102 is replaced with Thr;
  • Ala at position 101 is replaced with Asn and any amino acid except Pro is inserted between positions 101 and 102;
  • Leu at position 104 is replaced with Asn; Leu at position 104 is replaced with Asn and Thr at position 106 is replaced with Ser; Glu at position 105 is replaced with Asn and any amino acid except Pro is inserted between position 105 and 106;
  • Leu at position 107 is replaced with Asn; Leu at position 107 is replaced with Asn and Ser at position 109 is replaced with Thr;
  • Asp at position 108 is replaced with Asn and Leu at position 110 is replaced with Ser or Thr;
  • Glu at position 115 is replaced with Asn; Glu at position 115 is replaced with Asn and Ser at position 117 is replaced with Thr;
  • Gly at position 118 is replaced with Asn
  • Gly at position 118 is replaced with Asn and Ser at position 120 is replaced with Thr; Tyr at position 119 is replaced with Asn;
  • Tyr at position 119 is replaced with Asn and Thr at position 121 is replaced with Ser;
  • Ala at position 125 is replaced with Asn
  • Ala at position 125 is replaced with Asn and Ser at position 127 is replaced with Thr;
  • Gin at position 130 is replaced with Asn
  • Gin at position 130 is replaced with Asn and Ser at position 132 is replaced with Thr;
  • Gin at position 134 is replaced with Asn and Met at position 136 is replaced with Ser or Thr;
  • Asp at position 135 is replaced with Asn and Leu at position 137 is replaced with Ser or Thr;
  • Gin at position 139 is replaced with Asn and Asp at position 141 is replaced with Ser or Thr; Asp at position 141 is replaced with Asn; and
  • amino acid sequence of a protein of Formula I also has a modification selected from a second group consisting of:
  • Val at position 1 is deleted or is replaced with Glu, Asp, Lys, His, or Arg; Pro at position 2 is deleted or is replaced with Glu, Asp, Lys, His, or Arg; lie at position 3 is replaced with Glu, Asp, Lys, His, or Arg; Gin at position 4 is replaced with Asp, Glu, His,
  • Gin at position 7 is replaced with Asp, Glu, His, Lys, or Arg;
  • Asn at position 22 is replaced with Asp, Glu, His, Lys, or Arg, when position 24 is lie;
  • Thr at position 27 is replaced with Ala
  • Gin at position 28 is deleted or is replaced with Asp, Glu, His, Lys, or Arg;
  • Val at position 30 is replaced with Glu, Asp, Lys, His, or Arg;
  • Gin at position 34 is replaced with Asp, Glu, His, Lys, or Arg;
  • Val at position 36 is replaced with Glu, Asp, Lys, His, or Arg; Phe at position 41 is replaced with Glu, Asp, Lys,
  • His, or Arg lie at position 42 is replaced with Glu, Asp, Lys, His, or Arg;
  • Pro at position 43 is replaced with Glu, Asp, Lys, His, or Arg;
  • Leu at position 45 is replaced with Glu, Asp, Lys, His, or Arg;
  • His at position 46 is replaced with Glu, Asp, Lys, or Arg; Pro at position 47 is replaced with Glu, Asp, Lys,
  • His, or Arg lie at position 48 is replaced with Glu, Asp, Lys, His, or Arg;
  • Leu at position 49 is replaced with Glu, Asp, Lys, His, or Arg;
  • Thr at position 50 is replaced with Glu, Asp, Ser, Lys, His, or Arg; Met at position 54 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly;
  • Gin at position 56 is replaced with Asp, Glu, His, Lys, or Arg; Gin at position 62 is replaced with Asp, Glu, His,
  • Gin at position 63 is replaced with Asp, Glu, His, Lys, or Arg;
  • Met at position 68 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly;
  • Asn at position 72 is replaced with Asp, Glu, His, Lys, or Arg, when position 74 is lie;
  • Val at position 73 is replaced with Met; lie at position 74 is replaced with Glu, Asp, Lys, His, or Arg;
  • Gin at position 75 is replaced with Asp, Glu, His, Lys, or Arg;
  • Asn at position 78 is replaced with Asp, Glu, His, Lys, or Arg, when position 80 is Leu; Asn at position 82 is replaced with Asp, Glu, His,
  • Val at position 89 is replaced with Glu, Asp, Lys, His, or Arg;
  • Phe at position 92 is replaced with Glu, Asp, Lys, His, or Arg;
  • His at position 97 is replace with Gin, Asn, Ala, Gly, Ser, or Pro;
  • Trp at position 100 is replaced with Ala, Glu,
  • Ala at position 101 is replaced with Ser, Asn, Gly, His, or, Thr, or Val; Ser at position 102 is replaced with Arg;
  • Gly at position 103 is replaced with Ala; Glu at position 105 is replaced with Glu; Thr at position 106 is replaced with Lys or Ser; Leu at position 107 is replaced with Pro; Asp at position 108 is replaced with Glu; Gly at position 111 is replaced with Asp;
  • Gin at position 130 is replaced with Asp, Glu, His, Lys, or Arg;
  • Gin at position 134 is replaced with Asp, Glu, His, Lys, or Arg;
  • Met at position 136 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Trp at position 138 is replaced with Ala, Glu,
  • Gin at position 139 is replaced with Asp, Glu, His, Lys, or Arg; Leu at position 142 is replaced with Glu, Asp,
  • an obesity protein analog of the present invention has no more than about 5 modifications, and more preferably, has 1, 2, 3 or 4 modifications, and most preferably has 1, 2, or 3 modifications.
  • the inserted amino acid or amino acids are naturally-occurring amino acids selected from the group consisting of Ala, Cys, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr. More preferably, the inserted amino acid or acids are selected from the group consisting of Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Met, Asn, Gin, Arg, Ser, Thr, Val, Trp, and Tyr.
  • the inserted amino acids are selected from the group consisting of Ala, Asp, Glu, Phe, Gly, His, lie, Lys, Leu, Asn, Gin, Arg, Ser, Thr, Val, and Tyr.
  • the most preferred amino acids for insertion immediately after Asn are Ala, Asn, Gly, lie, Ser, Thr, and Val .
  • a preferred group of obesity protein analogs of the present invention are the analogs of Formula I that have a single amino acid modification. Within this group are obesity protein analogs with the following modifications in Formula I :
  • He at position 24 is replaced with Ser or Thr or is deleted;
  • Thr at position 27 is replaced with Asn
  • Ser at position 29 is replaced with Asn; Asn is inserted between positions 29 and 30;
  • Val at position 30 is replaced with Asn
  • Lys at position 35 is replaced with Asn; He at position 48 is replaced with Asn;
  • Thr at position 50 is replaced with Asn
  • He at position 74 is replaced with Ser or Thr;
  • Gin at position 75 is replaced with Asn
  • Leu at position 80 is replaced with Ser or Thr;
  • Arg at position 84 is replaced with Ser or Thr; Ala at position 91 is replaced with Asn;
  • Another preferred group of obesity protein analogs are those with a glycosylation site having the sequence NXT. This group consists of analogs having at least one of the following modifications:
  • Asp at position 8 is replaced with Asn;
  • Thr at position 10 is replaced with Asn;
  • Gin at position 7 is replaced with Asn and Asp at position 9 is replaced with Thr;
  • Ser at position 29 is replaced with Asn and Ser at position 31 is replaced with Thr;
  • Val at position 30 is replaced with Asn and Ser at position 32 is replaced with Thr;
  • Gin at position 34 is replaced with Asn and Val at position 36 is replaced with Thr;
  • Thr at position 50 is replaced with Asn and Ser at position 52 is replaced with Thr;
  • Gin at position 56 is replaced with Asn and Leu at position 58 is replaced with Thr;
  • Gin at position 62 is replaced with Asn and He at position 64 is replaced with Thr;
  • Gin at position 63 is replaced with Asn and Leu at position 65 is replaced with Thr;
  • Leu at position 65 is replaced with Asn and Ser at position 67 is replaced with Thr;
  • Gin at position 75 is replaced with Asn and Ser at position 77 is replaced with Thr;
  • Asp at position 85 is replaced with Asn and Leu at position 87 is replaced with Thr;
  • Ala at position 91 is replaced with Asn and Ser at position 93 is replaced with Thr;
  • Ser at position 93 is replaced with Asn and Ser at position 95 is replaced with Thr;
  • Trp at position 100 is replaced with Asn and Ser at position 102 is replaced with Thr;
  • Leu at position 107 is replaced with Asn and Ser at position 109 is replaced with Thr; Asp at position 108 is replaced with Asn and Leu at position 110 is replaced with Thr;
  • Glu at position 115 is replaced with Asn and Ser at position 117 is replaced with Thr; Gly at position 118 is replaced with Asn and Ser at position 120 is replaced with Thr;
  • Ala at position 125 is replaced with Asn and Ser at position 127 is replaced with Thr;
  • Gin at position 130 is replaced with Asn and Ser at position 132 is replaced with Thr;
  • Gin at position 134 is replaced with Asn and Met at position 136 is replaced with Thr;
  • Asp at position 135 is replaced with Asn and Leu at position 137 is replaced with Thr; Gin at position 139 is replaced with Asn and Asp at position 141 is replaced with Thr; and
  • Asp at position 141 is replaced with Asn and Ser at position 143 is replaced with Thr.
  • Another preferred group consists of obesity protein analogs wherein the glycosylation site is within the first 101 amino acids from the N-terminus.
  • This group consists of analogs having at least one of the following modifications :
  • Gin at position 4 is replaced with Asn and Val at position 6 is replaced with Ser or Thr;
  • Gin at position 7 is replaced with Asn and Asp at position 9 is replaced with Ser or Thr;
  • Asp at position 9 is replaced with Asn and Lys at position 11 is replaced with Ser or Thr;
  • Thr at position 10 is replaced with Asn
  • Thr at position 10 is replaced with Asn and Thr at position 12 is replaced with Ser;
  • Asp at position 23 is deleted or is replaced with Asn, and in either case, Ser at position 25 is replaced with Thr;
  • He at position 24 is deleted or is replaced with Ser or Thr;
  • Ser at position 25 is replaced with Asn and Thr at position 27 is replaced with Ser;
  • Thr at position 27 is replaced with Asn
  • Thr at position 27 is replaced with Asn and Ser at position 29 is replaced with Thr;
  • Gin at position 28 is replaced with Asn and any amino acid except Pro is inserted between position 28 and position 29;
  • Gin at position 28 is replaced with Asn and Val at position 30 is replaced with Ser or Thr;
  • Val at position 30 is replaced with Asn
  • Val at position 30 is replaced with Asn and Ser at position 32 is replaced with Thr;
  • Gin at position 34 is replaced with Asn and Val at position 36 is deleted; Gin at position 34 is replaced with Asn and Val at position 36 is replaced with Ser or Thr;
  • Lys at position 35 is replaced with Asn
  • Lys at position 35 is replaced with Asn and Thr at position 37 is replaced with Ser; Asn is inserted between positions 35 and 36;
  • Asp at position 40 is replaced with Asn and He at position 42 is replaced with Ser or Thr;
  • He at position 48 is replaced with Asn and Thr at position 50 is replaced with Ser;
  • Thr at position 50 is replaced with Asn
  • Thr at position 50 is replaced with Asn and Ser at position 52 is replaced with Thr; Asp at position 55 is replaced with Asn;
  • Gin at position 56 is replaced with Asn and Leu at position 58 is replaced with Ser or Thr; Gin at position 62 is replaced with Asn and He at position 64 is replaced with Ser or Thr;
  • Gin at position 63 is replaced with Asn and Leu at position 65 is replaced with Ser or Thr;
  • He at position 64 is replaced with Asn; He at position 64 is replaced with Asn and Thr at position 66 is replaced with Ser;
  • Leu at position 65 is replaced with Asn
  • Leu at position 65 is replaced with Asn and Ser at position 67 is replaced with Thr; Met at position 68 is replaced with Asn and Pro at position 69 is replaced with any amino acid except Pro; Met at position 68 is replaced with Asn, Pro at position 69 is replaced with any amino acid except Pro, and Ser at position 70 is replaced with Thr;
  • He at position 74 is replaced with Ser or Thr; Gin at position 75 is replaced with Asn;
  • Gin at position 75 is replaced with Asn and Ser at position 77 is replaced with Thr;
  • Asp at position 79 is replaced with Asn and Glu at position 81 is replaced with Ser or Thr; Leu at position 80 is replaced with Ser or Thr;
  • Arg at position 84 is replaced with Ser or Thr;
  • Asp at position 85 is replaced with Asn and Leu at position 87 is replaced with Ser or Thr;
  • Ala at position 91 is replaced with Asn; Ala at position 91 is replaced with Asn and Ser at position 93 is replaced with Thr;
  • Ser at position 93 is replaced with Asn and Ser at position 95 is replaced with Thr; Trp at position 100 is replaced with Asn;
  • Trp at position 100 is replaced with Asn and Ser at position 102 is replaced with Thr;
  • Ala at position 101 is replaced with Asn and any amino acid except Pro is inserted between positions 101 and 102.
  • Another preferred group consists of obesity protein analogs wherein the glycosylation site is relatively accessible at the surface of the obesity protein analog for glycosylation.
  • This group consists of analogs having at least one of the following replacements:
  • Ser at position 25 is replaced with Asn and Thr at position 27 is replaced with Ser; Thr at position 27 is replaced with Asn; Thr at position 27 is replaced with Asn and Ser at position 29 is replaced with Thr;
  • Val at position 30 is replaced with Asn; Val at position 30 is replaced with Asn and Ser at position 32 is replaced with Thr; Lys at position 35 is replaced with Asn;
  • Lys at position 35 is replaced with Asn and Thr at position 37 is replaced with Ser;
  • Thr at position 50 is replaced with Asn; Thr at position 50 is replaced with Asn and Ser at position 52 is replaced with Thr;
  • He at position 74 is replaced with Ser or Thr; Gin at position 75 is replaced with Asn;
  • Gin at position 75 is replaced with Asn and Ser at position 77 is replaced with Thr;
  • Arg at position 84 is replaced with Ser or Thr; Ser at position 93 is replaced with Asn; Ser at position 93 is replaced with Asn and Ser at position 95 is replaced with Thr;
  • Trp at position 100 is replaced with Asn; Trp at position 100 is replaced with Asn and Ser at position 102 is replaced with Thr; Leu at position 104 is replaced with Asn;
  • Leu at position 104 is replaced with Asn and Thr at position 106 is replaced with Ser;
  • Leu at position 107 is replaced with Asn; Leu at position 107 is replaced with Asn and Ser at position 109 is replaced with Thr;
  • Glu at position 115 is replaced with Asn; Glu at position 115 is replaced with Asn and Ser at position 117 is replaced with Thr;
  • Gly at position 118 is replaced with Asn
  • Gly at position 118 is replaced with Asn and Ser at position 120 is replaced with Thr;
  • Asp at position 141 is replaced with Asn and Ser at position 143 is replaced with Thr.
  • glycosylation site is in the range from amino acid 25 to amino acid 35.
  • This group consists of analogs having one of the following modifications:
  • Ser at position 25 is replaced with Asn and Thr at position 27 is replaced with Ser;
  • Thr at position 27 is replaced with Asn
  • Thr at position 27 is replaced with Asn and Ser at position 29 is replaced with Thr;
  • Gin at position 28 is replaced with Asn and any amino acid except Pro is inserted between position 28 and position 29;
  • Gin at position 28 is replaced with Asn and Val at position 30 is replaced with Ser or Thr;
  • Gin at position 34 is replaced with Asn and Val at position 36 is deleted; Gin at position 34 is replaced with Asn and Val at position 36 is replaced with Ser or Thr;
  • Lys at position 35 is replaced with Asn
  • Lys at position 35 is replaced with Asn and Thr at position 37 is replaced with Ser.
  • a more preferred group of analogs having a glycosylation site in the range of amino acid 25 to amino acid 35 consists of analogs having one the following replacements:
  • Thr at position 27 is replaced with Asn
  • Thr at position 27 is replaced with Asn and Ser at position 29 is replaced with Thr; Ser at position 29 is replaced with Asn;
  • Ser at position 29 is replaced with Asn and Ser at position 31 is replaced with Thr;
  • Val at position 30 is replaced with Asn
  • Val at position 30 is replaced with Asn and Ser at position 32 is replaced with Thr;
  • Lys at position 35 is replaced with Asn
  • Lys at position 35 is replaced with Asn and Thr at position 37 is replaced with Ser.
  • An especially preferred group of obesity protein analogs having a glycosylation site in the range of amino acid 25 to amino acid 35 consists of analogs having one of the following replacements:
  • Ser at position 29 is replaced with Asn and Ser at position 31 is replaced with Thr;
  • Val at position 30 is replaced with Asn; Val at position 30 is replaced with Asn and Ser at position 32 is replaced with Thr; and
  • Lys at position 35 is replaced with Asn.
  • Another preferred group of obesity protein analogs has a consensus N-linked glycosylation site at one or more positions from position 27 through position 50.
  • Another preferred group of obesity protein analogs has a consensus N-linked glycosylation site at one or more positions from position 95 through position 105.
  • glycosylated obesity protein analogs are those biosynthesized in a mammalian cell capable of glycosylating at a consensus N-linked glycosylation site. It will be appreciated that the glycosylation of such biosynthesized proteins is likely to be heterogeneous as to the position glycosylated and the structures of the glycosyl moieties .
  • the person of skill in the art will be aware of glycosyl groups typically attached to glycosylated proteins expressed in mammalian cells [Wright, A. et al . , Trends in Biotechnol . 15:26-32 (1997); Hummel, M. , et al., Eur. J. Biochem.
  • More preferred glycosylated obesity proteins of the present invention are those wherein the glycosyl structure is branched.
  • a highly preferred group consists of glycosylated obesity proteins of the present invention that have sialic acid residues at the terminus of each branch of the glycosyl structure .
  • Another preferred group consists of glycosylated obesity proteins of the present invention biosynthesized in a yeast cell or in an insect cell capable of glycosylating at a consensus N-linked glycosylation site.
  • the person of skill in the art will be aware of the "high mannose" glycosyl groups typically attached to glycosylated proteins expressed in yeast cells [Tanner, W., et al . , Biochim. Biophys . Acta 906:81-99 (1987); the entire disclosure of this reference relating to glycosyl structure is incorporated herein by reference] or in insect cells.
  • the obesity protein analog is preferably glycosylated by in vivo biosynthesis in an organism that is transformed and regulated to express a glycosylated recombinant protein.
  • Such in vivo biosynthesis includes producing the glycosylated protein in a transgenic animal or in a cell line or lines used for gene therapy in humans.
  • the obesity protein analog may alternatively be glycosylated using in vitro methods, such as, cell-free, enzyme-catalyzed methods employing the appropriate carbohydrate substrate or substrates and a glycosyltransferase activity or activities [Elbein, A. D., Trends in Biotechnol . 9:346-352 (1991)], or by chemical glycosylation.
  • Chemical and enzymatic methods of glycosylating proteins using a variety of activating groups are well-known [Baudys, M., et al., supra; Fu, M.-X., et al . , Diabetes 43:676 (1994); WO87/05330; Federoff, H. J. , et al . , Diabetes 42:509 (1993); Brownlee, M. , et al.,
  • a glycosyl group may be attached to the functional group nitrogen of a lysine, or to the amino nitrogen of the N-terminal amino acid.
  • the preferred method for preparing the obesity protein analogs and glycosylated obesity protein analogs of the present invention comprises biosynthesis using recombinant technology.
  • preparation of a protein by recombinant technology includes the steps of : a) constructing a synthetic or semi-synthetic DNA encoding the protein of interest; b) integrating said DNA into an expression vector in a manner suitable for the expression of the protein of interest, either alone or as a fusion protein; c) transforming or transfecting an appropriate eukaryotic or prokaryotic host cell with said expression vector; d) culturing said transformed or transfected host cell in a manner to express the protein of interest; and e) recovering and purifying the recombinantly produced protein of interest .
  • This invention provides DNA polynucleotide compounds encoding the obesity protein analogs of Formula I .
  • Those nucleic acid compounds which perform substantially the same function, in substantially the same manner, and result in the expression of an obesity protein analog of the present invention, as do the exemplified nucleic acid compounds, are encompassed within the present invention.
  • the protein compounds of the invention can be encoded by a multitude of different DNA polynucleotide sequences because most of the amino acids are encoded by more than one nucleic acid triplet due to the degeneracy of the amino acid code. Because these alternative nucleic acid sequences would encode the same amino acid sequences, the present invention further comprises these alternate nucleic acid sequences .
  • Synthetic DNA polynucleotide compounds encoding the human obesity protein may be designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into expression and amplification plasmids.
  • the restriction sites are chosen so as to properly orient the coding sequence of the target enzyme with control sequences to achieve proper in-frame reading and expression of the obesity protein analog molecule.
  • a variety of other such cleavage sites may be incorporated depending on the particular plasmid constructs employed and may be generated by techniques well- known in the art .
  • the desired DNA sequences can be generated using the polymerase chain reaction as described in U.S. Patent No. 4,889,818, which is herein incorporated by reference.
  • the template for the reaction can be a cDNA library (commercially available from Clonetech or Stratagene) or mRNA isolated from an appropriate adipose tissue.
  • cDNA library commercially available from Clonetech or Stratagene
  • mRNA isolated from an appropriate adipose tissue Such methodologies are well- known in the art [Sambrook, J., et al., supra . ] .
  • DNA encoding the obesity protein analogs of the present invention is preferably biosynthesized by mutating the DNA sequence that encodes human obesity protein, and amplifying the mutated DNA.
  • the appropriate mutations are made on the basis of the modification or modifications in the amino acid sequence that are desired, and on the basis of the known degeneracy of the genetic code.
  • the mutations are effected by any means known in the art, such as, for example, and without limitation, site-directed mutagenesis or ligation of the appropriate sequence into the DNA encoding the desired protein [Sambrook, J. , et al . , supra . ] .
  • Oligonucleotide mutagenesis is particularly well- suited for making amino acid replacements and deletions, and it is the preferred method for preparing the analogs of the present invention [Adelman, et al . , DNA 2:183 (1983)]. Oligonucleotides are readily synthesized using techniques well-known in the art [Crea, et al . , Proc . Nat ' l Acad. Sci . USA 75:5765 (1978)] . The DNA sequence may also be generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers [Applied Biosystems, Inc., Foster City, CA] .
  • PCR mutagenesis is also suitable for preparing DNA polynucleotide compounds encoding the analogs of the present invention [U.S. Patent No. 4,683,195, issued 28 Jul 1987; Current Protocols in Molecular Biology, Ausubel, et al . , eds., Greene Publishing Associates and Wiley-Interscience, volume 2, chapter 15 (1991)].
  • Mutants with more than one amino acid modification may be synthesized in any one of several ways. If the amino acid modifications are located close together in the amino acid sequence, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid modifications. If, however, the sites at which modifications are desired are more than about ten amino acids apart, one of two alternate methods may be used. In the first method, a separate oligonucleotide is generated for each amino acid to be modified. These oligonucleotides are annealed to the single-stranded template DNA simultaneously. The complementary, second strand of DNA that is synthesized from the template will then encode all of the desired amino acid modifications. The second method involves -repetitive rounds of mutagenesis. The constructed or isolated DNA sequences are useful for expressing the obesity protein analog either by direct expression or as a fusion protein.
  • Another method for making mutations in the DNA sequence encoding the human obesity protein sequence, or a variant known in the art is to cleave the DNA sequence at the appropriate position by digesting with a restriction enzyme or enzymes, recovering the properly cleaved DNA, synthesizing an oligonucleotide encoding the desired amino acid sequence for the amino acid modifications and flanking regions, such as, polylinkers with blunt ends, or digesting with the restriction enzymes used to cleave the DNA, thereby producing cohesive termini, and finally, ligating the synthetic DNA into the remainder of the obesity protein structural gene .
  • This invention encompasses recombinant DNA cloning vectors and expression vectors comprising the DNA polynucleotide compounds of the present invention.
  • the vectors are prepared using standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors.
  • To express a desired protein one inserts the engineered synthetic DNA sequence in any of a plethora of appropriate recombinant DNA expression vectors through the use of appropriate restriction endonucleases .
  • a synthetic coding sequence may be designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into these expression and amplification and expression plasmids.
  • Isolated cDNA coding sequences may be readily modified by the use of synthetic linkers to facilitate the incorporation of these sequences into the desired cloning vectors by techniques well-known in the art.
  • the particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed.
  • the restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in-frame reading and expression of the protein.
  • the desired coding sequence is inserted into an expression vector in the proper orientation to be transcribed from a promoter and ribosome binding site, both of which should be functional in the host cell in which the protein is to be expressed.
  • An example of such an expression vector is a plasmid described in Belagaje et al . , U.S. patent No. 5,304,473, the teachings of which are herein incorporated by reference.
  • the gene encoding A-C-B proinsulin described in U.S. Patent No. 5,304,473 can be removed from the plasmid pRB182 with restriction enzymes
  • Ndel and BamHI The isolated coding sequences of DNA can be inserted into the plasmid backbone on a Ndel/BamHI restriction fragment cassette.
  • Plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used with these host cells.
  • the vector usually has a replication site, marker genes that provide phenotypic selection in transformed cells, one or more promoters, and a polylinker region containing several restriction sites for insertion of foreign DNA.
  • Plasmids typically used for transformation of E. coli include pBR322, pUCl ⁇ , pUC19, pUCll ⁇ , pUC119, and Bluescript M13 , all of which are described in sections 1.12-1.20 of Sambrook, et al . supra .
  • E. coli is typically transformed using pBR322, a plasmid derived from an E.
  • Plasmid pBR322 contains genes for ampicillin (amp) and tetracycline resistance (tet) and thus provides easy means for identifying transformed cells.
  • the pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA technology. Promoters suitable for use with prokaryotic hosts include: the ⁇ -lactamase promoter, which is available on a plasmid vector that also provides the replicon and ⁇ - lactamase gene (pGX2907, ATCC No.
  • the lactose promoter system [Chang et al . , Nature 275:615 (1978); Goeddel et al . , Nature 281:544 (1979)]; the alkaline phosphatase promoter; the tryptophan (trp) promoter system, which is provided on the plasmid vector pATHl (ATCC No. 37,695) and that is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter; and hybrid promoters, such as, the tac promoter, which may be isolated from plasmid pDR540 (ATCC No. 37,282) .
  • the DNA polynucleotide compounds are preferably integrated into vectors suitable for eukaryotic expression systems .
  • Plasmids constructed for expression of the proteins of the present invention in mammalian and other eukaryotic host cells can utilize a wide variety of promoters.
  • the present invention is in no way limited to the use of the particular promoters exemplified herein.
  • Promoters such as the SV40 late promoter, promoters from eukaryotic genes, such as, for example, the estrogen- i ducible chicken ovalbumin gene, the interferon genes, the gluco-corticoid-inducible tyrosine aminotransferase gene, and the thymidine kinase gene, and the major early and late adenovirus genes can be readily isolated and modified to express the genes of the present invention.
  • Eukaryotic promoters can also be used in tandem to drive expression of a coding sequence of this invention.
  • retroviruses are known that infect a wide range of eukaryotic host cells. The long terminal repeats in the retroviral DNA frequently encode functional promoters and, therefore, may be used to drive expression of the nucleic acids of the present invention.
  • Preferred promoters controlling transcription in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40) , adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e . g. , the ⁇ -actin promoter.
  • viruses such as: polyoma, Simian Virus 40 (SV40) , adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e . g. , the ⁇ -actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication [Fiers, et al . , Nature, 273:113 (1978)].
  • the immediate early promoter of the human cytomegalovirus may be obtained from plasmid pCMBb (ATCC No. 77,177). Of course, promoters from the host cell or related species also are useful herein.
  • Plasmid pRSVcat (ATCC No. 37,152) comprises portions of a long terminal repeat of the Rous Sarcoma virus, a virus known to infect chickens and other host cells. This long terminal repeat contains a promoter which is suitable for use in the vectors of this invention [Gorman, H., et al., Proc. Nat 'l Acad. Sci . USA 79:6777 (1982)].
  • the plasmid pMSVi (NRRL B-15929) comprises the long terminal repeats of the Murine Sarcoma virus, a virus known to infect mouse and other host cells .
  • the mouse metallothionein promoter has also been well characterized for use in eukaryotic host cells and is suitable for use in the expression of the nucleic acids of the present invention.
  • the mouse metallothionein promoter is present in the plasmid pdBPV-MMTneo (ATCC No. 37,224) which can serve as the starting material of other plasmids of the present invention.
  • Enhancers are cis-acting elements of DNA, usually about 10- 300 bp, that act on a promoter to increase its transcription. Enhancers are relatively oriented and positioned independently and have been found 5' [Laimins, L. et al . , Proc . Nat ' l Acad . Sci . USA 78:993 (1981)] and 3"
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding protein. The 3 ' -untranslated regions also include transcription termination sites.
  • Expression vectors may contain a selection gene, also termed a selectable marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR) , which may be derived from the Bglll/Hindlll restriction fragment of pJOD-10 (ATCC No.
  • DHFR dihydrofolate reductase
  • thymidine kinase such as the herpes simplex virus thymidine kinase that is contained on the BamHI fragment of the vP-5 clone (ATCC No. 2028), or neomycin (G418) resistance genes that are obtainable from the pNN414 yeast artificial chromosome vector (ATCC No. 37,682).
  • selectable markers When such selectable markers are successfully transferred into a mammalian host cell, the transfected mammalian host cell can survive if placed under selective pressure.
  • the first strategy is based on a cell's metabolism and the use of a mutant cell line that lacks the ability to grow without a supplemented media.
  • Two examples are: CHO DHFR- cells (ATCC CRL-9096) and mouse LTK " cells (L-M(TK-) ATCC CCL-2.3). Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in unsupplemented media.
  • the second strategy is dominant selection, which may be used in any cell type and does not require the use of a mutant cell line.
  • a drug is used to arrest growth of a host cell. Cells in the population that have a gene expresses a protein conveying drug resistance survive the selection. Examples of such dominant selection use the drugs neomycin [Southern, P. et al . , J. Molec. Appl . Genet . 1:327 (1982)], mycophenolic acid [Mulligan, R. C. et al., Science 209:1422 (1980)], or hygromycin [Sugden, B., et al . , Mol Cell . Biol .
  • a preferred vector for eukaryotic expression is pRc/CMV.
  • pRc/CMV is commercially available from Invitrogen Corporation, 3985 Sorrento Valley Blvd., San Diego, CA 92121.
  • the ligation mixtures are used to transform E. coli K12, strain DHIOB (ATCC No. 31,446; also available from Gibco/BRL in competent form) and successful transformants are selected by antibiotic resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequenced [Messing, et al . , Nucleic Acids Res . 9:309 (1981)] .
  • the plasmid YRp7 is commonly used (ATCC No. 40,053) [Stinchcomb, et al . ,
  • This plasmid already contains the trp gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, (ATCC No. 44,076 or PEP4-1) [Jones, Genetics 85:12 (1977)] .
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase, which is found on plasmid pAP12BD (ATCC No. 53,231) [U.S. Patent No. 4,935,350, issued 19 Jun 1990], or other glycolytic enzymes such as enolase, which is found on plasmid pACl (ATCC No. 39,532), glyceraldehyde-3 -phosphate dehydrogenase , which is derived from plasmid pHcGAPCl (ATCC Nos. 57,090, 57,091), zymomonas mobilis (U.S. Patent No.
  • yeast promoters which contain inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase II, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, which is contained on plasmid vector pCL28XhoLHBPV (ATCC No. 39,475) [U.S. Patent No. 4,840,896], glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization, such as, GAL1, which is found on plasmid pRY121 (ATCC No. 37,658) .
  • Yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae, found in conjunction with the CYC1 promoter on plasmid YEpsec--hIlbeta (ATCC No. 67,024), also are advantageously used with yeast promoters .
  • the pSV2-type vectors comprise segments of the simian virus 40 (SV40) genome that constitute a defined eukaryotic transcription unit-promoter, intervening sequence, and polyadenylation site.
  • the plasmid pSV2-type vectors transform mammalian and other eukaryotic host cells by integrating into the host cell chromosomal DNA.
  • a large number of plasmid pSV2-type vectors have been constructed, such as plasmid pSV2-gpt, pSV2-neo, pSV2-dhfr, pSV2-hyg, and pSV2- ⁇ - globin, in which the SV40 promoter drives transcription of an inserted gene.
  • These vectors are suitable for use with the coding sequences of the present invention and are widely available from sources such as the ATCC or the Northern Regional Research Laboratory (NRRL) , 1815 N. University Street, Peoria, Illinois, 61604.
  • the plasmid pSV2-dhfr (ATCC 37146) comprises a murine dihydrofolate reductase (dhfr) gene under the control of the SV40 early promoter. Under the appropriate conditions, the dhfr gene is known to be amplified, or copied, in the host chromosome. This amplification can result in the amplification of closely-associated DNA sequences and can, therefore, be used to increase production of a protein of interest [Schimke, J., Cell , 35:705-713 (1984)] .
  • dhfr murine dihydrofolate reductase
  • An especially preferred expression vector system employs one of a series of vectors containing the BK enhancer, an enhancer derived from the BK virus, a human papovavirus .
  • the most preferred such vector systems are those which employ not only the BK enhancer but also the adenovirus-2-early region IA (E1A) gene products.
  • the E1A gene products are immediate-early gene products of adenovirus , a large DNA virus .
  • a preferred expression vector employed in the present invention is the phd series of vectors which comprise a BK enhancer in tandem with the adenovirus late promoter to drive expression of useful products in eukaryotic host cells.
  • the construction and method of using the phd plasmid, as well as related plasmids, are described in U.S. Patents 5,242,688, issued September 7, 1993, and 4,992,373, issued February 12, 1991, both of which are herein incorporated by reference.
  • Escherichia coli K12 GM48 cells harboring the plasmid phd are available as part of the permanent stock collection of the Northern Regional Research Laboratory under accession number NRRL B-18525. The plasmid may be isolated from this culture using standard techniques.
  • the plasmid phd contains a unique Bell site which may be utilized for the insertion of the gene encoding the protein of interest.
  • linkers or adapters may be employed in cloning the gene of interest into this BclT site.
  • the phd series of plasmids functions most efficiently when introduced into a host cell which produces the E1A gene product, cell lines such as AV12-664, 293 cells, and others, described supra .
  • viruses are also appropriate vectors.
  • the adenovirus, the adeno-associated virus, the vaccinia virus, the herpes virus, the baculovirus, the Simliki Forest Virus, and the rous sarcoma virus are useful.
  • Such a method is described in U.S. Patent 4,775,624, herein incorporated by reference.
  • Several alternate methods of expression are described in Sambrook, et al . , supra, at 16.3-17.44.
  • Yet another embodiment of the invention is a method of using DNA polynucleotide compounds comprising segments that encode obesity protein analogs to transform a cell .
  • Transformation of the mammalian cells can be performed by any of the known processes including, but not limited to, the protoplast fusion method, the calcium phosphate co- precipitation method, electroporation and the like [Sambrook, J., et al . , supra, at 3:16.30-3:16.66].
  • Host cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes.
  • prokaryotic host cells are preferred for the initial cloning steps because they produce large amounts of DNA, and can produce single-stranded DNA templates for site- directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated.
  • Suitable prokaryotic host cells include E. coli K12 strain 294 (ATCC No. 31,446), E. coli W3110 (ATCC No. 27,325), E. coli K12 strain DHIOB (ATCC 31,446), E. coli X1776 (ATCC No. 31,537), and E.
  • E. coli B and numerous other E. coli strains, such as, HB101, JM101, NM522, NM538, NM539, L201, L687, L693, L507, L640, L641, and L695.
  • a preferred strain of E. coli employed in the cloning and expression of the DNA nucleotide sequences of this invention is RV308 (ATCC No. 31,608) [U.S. Patent No. 4,551,433, issued November 5, 1985] .
  • prokaryotic host cells including enterobacteriaceae, such as Bacillus subtilis, Salmonella typhimurium, or Serratia marcescans, and various pseudomonas species may also be used to clone the DNA polynucleotides of the invention and to express the obesity protein analogs of the invention.
  • enterobacteriaceae such as Bacillus subtilis, Salmonella typhimurium, or Serratia marcescans
  • various pseudomonas species may also be used to clone the DNA polynucleotides of the invention and to express the obesity protein analogs of the invention.
  • enterobacteriaceae such as Bacillus subtilis, Salmonella typhimurium, or Serratia marcescans
  • pseudomonas species may also be used to clone the DNA polynucleotides of the invention and to express the obesity protein analogs of the invention.
  • other bacteria especially S re omyces, spp., may be employed in the prokaryotic clon
  • the transformed cells are selected by growth on an antibiotic, such as tetracycline (tet) or ampicillin (amp) , to which the hosts are rendered resistant due to the presence of tet and/or amp resistance genes on the vector.
  • an antibiotic such as tetracycline (tet) or ampicillin (amp)
  • tet tetracycline
  • amp ampicillin
  • transformed cells may be selected by the DHFR/MTX system.
  • the transformed cells are grown in culture and the plasmid DNA is then isolated.
  • the plasmid DNA may be analyzed by either restriction mapping or DNA sequencing, or DNA sequencing, or both. These methods will be well-known to the person of skill in the art .
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the techniques of transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as Sambrook, et al., supra .
  • a protein of the present invention may be expressed without glycosylation in prokaryotic host cells and, after recovery of the protein, it may be glycosylated in vitro.
  • the preferred system for expressing the obesity protein analogs of the present invention is a eukaryotic host cell having the enzymatic systems for glycosylating the protein.
  • Such systems include: cultured eukaryotic microorganisms, for example, yeast; cultured eukaryotic cells from multicellular organisms, for example, plant cell culture, insect cell culture, mammalian cell culture; transgenic expression in an intact, non-human multicellular organism; and expression in the body of a human patient, aided by gene therapy.
  • prokaryotes are used for cloning of DNA sequences in constructing the vectors of this invention.
  • Prokaryotes may also be employed to express large quantities of the protein of interest.
  • the Escherichia coli K12 strain 294 ATCC No. 31446
  • Other strains of E. coli K12 which may be used (and their relevant genotypes) include the following.
  • JM109 recAl, el4 " (mcrA), supE44, endAl, hsdR17(r ⁇ _ , m ⁇ + ) , gyrA96, relAl, thi-1, E(lac-proAB) , F 1 [traD36, proAB+ lacl ⁇ l, lacZEM15]
  • strains are all commercially available from suppliers such as : Bethesda Research Laboratories (Gaithersburg, MD 20877) and Stratagene Cloning Systems (La Jolla, California 92037) or are readily available to the public from sources such as the American Type Culture Collection (12301 Parklawn Drive, Rockville, MD, 10852- 1776) . Except where otherwise noted, these bacterial strains can be used interchangeably.
  • the genotypes listed are illustrative of many of the desired characteristics for choosing a bacterial host and are not meant to limit the invention in any way. The genotype designations are in accordance with standard nomenclature [Sambrook, et al . , supra] .
  • RV308 [ATCC No. 31,608, U.S. Patent No. 4,551,433, issued November 5, 1985] .
  • Saccharomyces cerevisiae, or common baker's yeast is the most commonly used lower eukaryotic host microorganism.
  • Other useful lower eukaryotic host cells are Schizosaccharomyces pombe [Beach, et al . Nature 290:140 (1981)], Kluyveromyces species [U.S. Patent No. 4,943,529; U.S. Patent No. 5,612,029, which are incorporated herein by reference], Pichia pastoris [Sreekrishna, et al .
  • Candida species Trichoderma reesia, Neurospora crassa [Case, et al . , Proc . Nat ' l Acad. Sci . USA 76:5259-5263 (1979)], Schwanniomyces occidentalis, various filamentous fungi, and Aspergillus nidulans
  • the proteins of the present invention may also be produced in mammalian host cells.
  • the present invention is not limited to use in a particular host cell.
  • a variety of host cells are available from depositories such as the American Type Culture Collection (ATCC) and are suitable for use with the vectors of the present invention.
  • ATCC American Type Culture Collection
  • the choice of a particular host cell depends to some extent on the particular expression vector used to drive expression of the obesity protein analog-encoding nucleic acids of the present invention.
  • Exemplary mammalian host cells suitable for use in the present invention are listed in Table I.
  • Preferred mammalian host cells for expressing the vectors encoding the claimed proteins include: African green monkey kidney line transformed by SV40 (COS- 7, ATCC CRL-1651) , transformed human primary embryonal kidney cell line 293 [Graham, F. L. et al . , J. Gen Virol . 36:59-72 (1977)], baby hamster kidney cells BHK-21(C-13) (ATCC CCL-
  • mice Sertoli cells TM4, ATCC CRL-1715, African green monkey kidney cells (VERO 76, ATCC CRL-1587), human cervical epithelial carcinoma cells (HeLa, ATCC CCL-2) , canine kidney cells (MDCK, ATCC CCL-34) , buffalo rat liver cells (BRL 3A, ATCC CRL-1442) , human diploid lung cells (WI-38, ATCC CCL- 75), human hepatocellular carcinoma cells (Hep G2, ATCC HB- 8065), and mouse mammary tumor cells (MMT 060562, ATCC CCL51) .
  • mouse Sertoli cells TM4, ATCC CRL-1715, African green monkey kidney cells (VERO 76, ATCC CRL-1587), human cervical epithelial carcinoma cells (HeLa, ATCC CCL-2) , canine kidney cells (MDCK, ATCC CCL-34) , buffalo rat liver cells (BRL 3A, ATCC CRL-1442) , human diploid lung cells (WI-38, ATCC CCL
  • An especially preferred cell line employed in this invention is the widely available cell line AV12-664 ("AV12").
  • AV12 This cell line is available from the American Type Culture Collection under the accession number ATCC CRL 9595.
  • the AV12 cell line was constructed by injecting a Syrian hamster in the scruff of the neck with human adenovirus 12 and isolating cells from the resulting tumor.
  • a further embodiment of the invention consists of a method of using a host cell to express obesity protein analogs, and glycosylated obesity protein analogs.
  • a host cell either prokaryotic or eukaryotic, that has been transformed, is cultured in an appropriate medium until a substantial cell mass has been obtained.
  • the cloned DNA polynucleotides encoding the obesity protein analogs of the present invention may also be employed in the production of transgenic animals in which expression or over-expression of the proteins of the present invention can be assessed. These DNA polynucleotides may also be used to construct "knockout" animals in which the expression of the native cognate of the gene is suppressed.
  • thermo-inducible promoter-operator regions such as the cl857 thermo-inducible lambda-phage promoter-operator region, require a temperature shift from about 30°C to about
  • the transformed host cells are plated on appropriate media under the selective pressure of the antibiotic corresponding to the resistance gene present on the expression plasmid. The cultures are then incubated for a time and temperature appropriate to the host cell line employed. Transformants are then selected.
  • the proteins of this invention may be synthesized either by direct expression or as a fusion protein.
  • a fusion protein comprises the protein of interest as a translational fusion with a protein or with a peptide.
  • the protein or peptide is removed either in vivo during the post-translational processing of the expressed protein, or in vitro by enzymatic or chemical cleavage. It is often observed in the production of certain proteins in recombinant systems that expression as a fusion protein is less detrimental to host cell viability, increases the yield of the desired peptide, or provides a convenient means of purifying the protein of interest. When the sequences are used in a fusion gene, the resulting product will require enzymatic or chemical cleavage.
  • peptidases which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known. Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites.
  • chemicals e.g. cyanogen bromide
  • the skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites [U.S.
  • Proteins that are expressed in high-level bacterial expression systems characteristically aggregate in granules (inclusion bodies) which contain high levels of the over-expressed protein. Such protein granules must be solubilized before the expressed protein can be isolated and purified.
  • a variety of techniques using strongly denaturing solutions such as guanidinium-HCl and/or weakly denaturing solutions such as urea are used to solubilize the proteins. Removal or dilution of the denaturing agents allows the denatured protein to assume its native conformation. The particular conditions for denaturation and folding are determined by the particular protein expression system and the protein in question.
  • the present invention provides a method for treating obesity.
  • the method comprises administering to an organism an effective amount of a glycosylated obesity protein or a glycosylated obesity protein analog.
  • Effective doses in the range of about 1 ⁇ g/kg to about 1000 ⁇ g/kg.
  • a preferred dose is from about 10 to 100 ⁇ g/kg of active compound.
  • a typical daily dose for an adult human is from about 0.05 to about 100 mg.
  • the glycosylated proteins can be administered in a single daily dose or in multiple doses per day.
  • the treatment regime may require administration over extended periods of time.
  • the amount per administered dose or the total amount administered will be determined by the physician, and will depend on such factors as the nature and severity of the disease, the age and general health of the patient and the tolerance of the patient to the compound.
  • the present invention further provides pharmaceutical formulations comprising the glycosylated protein compounds of the present invention.
  • the glycosylated proteins preferably in the form of a pharmaceutically acceptable salt, can be formulated for parenteral administration for the therapeutic or prophylactic treatment of obesity.
  • the glycosylated proteins can be admixed with conventional pharmaceutical carriers and excipients.
  • the concentration of glycosylated protein in the formulation is preferably about from about 0.5 mg/mL to about 100 mg/mL. More preferably, the concentration of obesity protein analog in the formulation is from about 0.5 mg/mL to about 50 mg/mL. Still more preferably, the concentration of obesity protein analog in the formulation is from about 1 mg/mL to about 25 mg/mL.
  • the concentration of obesity protein analog in the formulation is from about 1 mg/mL to about 10 mg/mL.
  • Other preferred ranges of concentration of obesity protein analog in the formulation are from about 0.5 mg/mL to about 20 mg/mL, from 0.5 mg/mL to about 5 mg/mL, and from about 2 mg/mL to about 20 mg/mL.
  • the present glycosylated proteins may be administered alone or in combination with other anti- obesity agents or agents useful in treating diabetes. Peripheral, parenteral administration is preferred.
  • the formulations prepared in accordance with the present invention may be administered using a syringe, injector, pump, or any other device recognized in the art for parenteral administration.
  • the amount of a formulation of the present invention that would be administered to treat obesity will depend on a number of factors, among which are included, without limitation, the patient's sex, weight and age, the underlying causes of obesity, the route of administration and bioavailability, the persistence of administered obesity protein analog in the body, the formulation, and the potency of the obesity protein analog.
  • the amount per administration should also take into account the interval between doses, and the bioavailability of the obesity protein analog from the formulation.
  • Administration of the formulation of the present invention could be continuous. It is within the skill of the ordinary physician to titrate the dose and rate or frequency of administration of the formulation of the present invention to achieve the desired clinical result.
  • Another preferred route of administration of the obesity protein analogs, glycosylated obesity proteins and glycosylated obesity protein analogs is by pulmonary administration.
  • the obesity protein analogs, glycosylated obesity proteins and glycosylated obesity protein analogs of the present invention can be delivered by any of a variety of inhalation devices known in the art for administration of a therapeutic agent by inhalation, wherein the therapeutic agent is deposited into the lower airways, primarily into the alveoli.
  • inhalation devices known in the art for administration of a therapeutic agent by inhalation, wherein the therapeutic agent is deposited into the lower airways, primarily into the alveoli.
  • Those skilled in the art of pulmonary administration will recognize that the formulation of such proteins, the quantity of the formulation delivered, and the duration of administration will depend on the type of inhalation device employed.
  • the frequency of administration and length of time for which the system is activated will depend on the concentration of protein agent in the aerosol. Shorter periods of administration can be used at higher concentrations of protein agents in the nebulizer solution. Devices such as metered dose inhalers can produce higher aerosol concentrations, and can be operated for shorter periods to deliver the desired amount of protein agent.
  • MMAD mass median aerodynamic diameter
  • the formulation of obesity protein analogs, glycosylated obesity proteins and glycosylated obesity protein analogs for pulmonary administration is selected to yield the desired protein size in the chosen inhalation device.
  • the powder can include a bulking agent, a carrier, or an excipient.
  • Such additives are included in a dry powder formulation of obesity protein analogs, glycosylated obesity proteins and glycosylated obesity protein analogs, for example, to facilitate processing of the formulation, to provide advantageous powder properties, to facilitate dispersion of the powder from the inhalation device, or to stabilize the formulation.
  • the additive will not irritate the patient's airways.
  • Such additives include, without limitation, mono-, di-, and polysaccharides, sugar alcohols, and other polyols, such as, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch,, or combinations thereof.
  • surfactants such as, sorbitols, diphosphatidyl choline, or lechithin, and the like are used.
  • Formulations for administration as an aerosol spray or via a nebulizer are comprised of liquid formulations of the obesity protein analogs, glycosylated obesity proteins and glycosylated obesity protein analogs of the present invention, and may include, an isotonicity agent, a buffer, a preservative, a surfactant, or a metal.
  • Such formulations may also contain an excipient or an agent for stabilizing the formulation, such as, a buffer, a reducing agent, a bulk protein, or a carbohydrate.
  • Bulk proteins useful in such formulations include serum albumin.
  • Carbohydrates used to stabilize such formulations of obesity protein analogs, glycosylated obesity proteins and glycosylated obesity protein analogs include, sucrose, mannitol, lactose, trehalose, glucose, and the like.
  • Various conventional surfactants can be used to stabilize the formulation, such as, polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol esters. Amounts will generally range between 0.001% and 4% by weight of the formulation.
  • Especially preferred surfactants for stabilizing formulations of obesity protein analogs, glycosylated obesity proteins and glycosylated obesity protein analogs of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like.
  • the pH of the formulations of the present invention is from about 4.0 to about 8.0.
  • the formulations of the present invention have pH between about 6.0 and about 8.0, and most preferably from about 6.8 to about 8.0.
  • Glycerin is the preferred isotonicity agent.
  • concentration of the isotonicity agent is in the range known in the art for parenteral formulations, and for glycerin, is preferably about 16 mg/mL to about 25 mg/mL.
  • the protein is administered in commonly used intravenous fluid (s) and administered by infusion.
  • Such fluids for example, physiological saline, Ringer's solution or 5% dextrose solution can be used.
  • the most effective preservatives, phenol and m- cresol, or mixtures thereof, can cause protein aggregation.
  • concentration of preservative in the formulations of the present invention is that required to maintain preservative effectiveness.
  • the relative amounts of preservative necessary to maintain preservative effectiveness varies with the preservative used. Generally, the amount necessary can be found in See, e.g., WALLHAUSER, K.DH., DEVELOP. BIOL. STANDARD. 24, pp. 9-28 (Basel, S. Krager, 1974) .
  • the optimal concentration of the preservative depends on the preservative, its solubility, and the pH of the formulation.
  • the preferred preservatives are benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, ethyl paraben, chlorobutanol , o- cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal and various mixtures thereof. More preferred preservatives are phenol, m-cresol, benzyl alcohol, and methylparaben.
  • additives such as a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate) , Tween 40 (polyoxyethylene (20) sorbitan monopalmitate) , Tween 80 (polyoxyethylene (20) sorbitan monooleate) , Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers) , and PEG (polyethylene glycol) may optionally be added to the formulation to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation and may reduce protein aggregation.
  • solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate) , Tween 40 (polyoxyethylene (20) sorbitan monopalmitate) , Tween 80 (polyoxyethylene (20) sorbitan monooleate) , Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers)
  • a sterile formulation preferably a suitable soluble salt form of a glycosylated protein of this invention, for example the hydrochloride salt, can be dissolved and administered in a pharmaceutical diluent such as pyrogen-free water
  • a suitable insoluble form of a glycosylated protein of this invention may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e . g. , an ester of a long chain fatty acid such as ethyl oleate .
  • the formulations of the present invention can be prepared using conventional dissolution and mixing procedures.
  • a suitable formulation for example, a measured amount of obesity protein analog or a glycosylated obesity protein in water is combined with the desired preservative in water in quantities sufficient to provide the protein and preservative at the desired concentration.
  • the pH of the formulation may be adjusted either before or after combining the obesity protein analog and the preservative.
  • the formulation is generally sterile filtered prior to administration. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, the timing of pH adjustment, the surfactant used, if any, the temperature and ionic strength at which the formulation is prepared, may be optimized for the concentration and means of administration used.
  • Plasmid pOJ726 Plasmid constructions were carried out using standard methodology as described by Sambrook, J., et al . , supra, the teaching of which is incorporated herein by reference. Enzymes and other reagents used for plasmid constructions were obtained from New England Biolabs, (NEB, Beverly MA) , Ambion (Austin, TX) , Amersham (Arlington Heights, IL) , Novex (San Diego, CA) and Gibco BRL (Bethesda, MD) .
  • the starting plasmid contains DNA that encodes an analog of human obesity protein consisting of human obesity protein having the dipeptide, Met-Arg, appended to the N-terminus.
  • Plasmid pOJ722 was digested with Ndel and Ba HI and the small 450 bp fragment was ligated into pUC19 (NEB) , which had been linearized with Ndel and BamHI.
  • the ligation products were transformed into DHIOB cells (ATCC No. 31,446; also available from Gibco/BRL in competent form) and transformants were plated on tryptone yeast agar medium containing 100 ⁇ g/ml ampicillin (Sigma) .
  • Plasmid pOJ834 Plasmid pOJ726 was further modified to encode an obesity protein analog that can be produced in eukaryotic hosts and secreted into the culture medium.
  • a DNA polynucleotide sequence encoding a secretion signal peptide sequence was added so that the following amino acid sequence would be attached at the amino-terminal end of the obesity protein analog: 1 5 10 15
  • the DNA sequence for this signal peptide is identical to that of the native human obesity protein sequence.
  • plasmid pOJ726 was digested with Ndel and Kpnl such that the signal peptide sequence could be ligated between these two sites.
  • the linearized 4kb Ndel to Kpnl fragment was gel purified and ligated with a chemically synthesized double-stranded D ⁇ A fragment (obtained by annealing two complementary single-stranded oligonucleotides containing NdeJ and Kpnl compatible ends) encoding the signal sequence.
  • the ligation products were transformed into DHIOB cells and transformants were plated on tryptone yeast agar medium containing 100 ⁇ g/ml ampicillin (Sigma) . Colonies which grew at 37°C were picked and inoculated into tryptone yeast broth containing 100 ⁇ g/ml ampicillin and grown overnight at 37 C. Plasmid D ⁇ A from these cultures were isolated for further analysis. Plasmids with the expected structure as confirmed by restriction analysis and D ⁇ A sequencing were designated pOJ834.
  • Plasmids pOJ841-850 Consensus ⁇ -linked glycosylation sites were introduced at four different positions. The original sequences, the mutations and the corresponding plasmid designations are summarized in the table below:
  • pOJ845/pOJ850 were introduced into pOJ834 by digesting pOJ834 with EcoRV and Bglll and gel purifying the resulting linear fragment.
  • Chemically synthesized double- stranded DNA fragments obtained by annealing two complementary single-stranded oligonucleotides encoding the potential glycosylation sites were ligated between the EcoRV and Bglll sites.
  • sequences of the chemically-synthesized DNA fragments are shown below.
  • the sequences that are shown correspond to one of the two chemically-synthesized oligonucleotides.
  • the complementary strands are not shown but can be easily obtained by using a computer program, such as the Wisconsin Package (Genetics Computer Group, Madison, WI, GCG) .
  • the following DNA sequence encodes amino acids 24-
  • the following DNA sequence encodes amino acids 24-62, wherein lysine at position 35 is replaced with asparagine :
  • DNA sequence encodes amino acids 24- 62, wherein threonine at position 50 is replaced with asparagine : ATCTCACACA CACAGTCAGT ctcgagcAAA CAGAAAGTCA CAGGCTTGGA CTTCATACCT 60 GGGCTGCACC CCATACTGaa cTTGTCTAAA ATGGACCAGA CACTGGCAGT CTATCAACA 119 (SEQ ID N0:6) .
  • DNA sequence encodes amino acids 24- 62, wherein threonine at position 27 is replaced with asparagine , serine at position 29 is replaced with asparagine, lysine at position 35 is replaced with asparagine , and threonine at position 50 is replaced with asparagine :
  • GGGCTGCACC CCATACTGaa cTTGTCTAAA ATGGACCAGA CACTGGCAG TCTATCAACA 119 SEQ ID NO: 7 .
  • the ligation mix was transformed into DH10B cells and plated on tryptone yeast agar plates containing 100 ⁇ g/ml ampicillin. Colonies that grew up overnight at 37°C were picked and inoculated into tryptone yeast broth containing 100 ⁇ g/ml ampicillin and grown up at 37 C.
  • Plasmid DNA was isolated from these cultures and plasmids containing the correct sequence were identified by restriction analysis and DNA sequencing.
  • the gene sequences encoding the mutated anti- obesity proteins were subcloned into pSFVl by digesting pOJ841-pOJ845 with BamHI and ligating into BamHI-digested pSFVl. Prior to ligation, the BamHJ-digested pSFVl was treated with bacterial alkaline phosphatase to prevent recircularization.
  • the ligation mix was transformed into DH10B cells and plated on tryptone yeast agar plates containing 100 ⁇ g/ml ampicillin.
  • Plasmid DNA was isolated from these cultures and plasmids containing the correct sequence were identified by restriction analysis and DNA sequencing. The resulting plasmids were designated pOJ846-pOJ850, as indicated in the table above.
  • SFV Gene Expression System from Gibco-BRL (Bethesda, MD) that employed a Simliki Forest Virus (SFV) helper plasmid.
  • the plasmids pOJ846-pOJ850 and the pSFV2 helper plasmid DNA were linearized by Spel digestion, followed by phenol extraction according to the protocol in the Gibco SFV Gene Expression System manual.
  • the linearized DNAs were transcribed into 5-capped RNAs according to the protocol in Ambion's mMessage mMachine SP6 RNA transcription kit (Ambion, Inc., Austin, TX) , which is herein incorporated by reference.
  • RNAs and the pSFV2 helper plasmid RNA were then transfected into Baby Hamster Kidney cells (BHK, ATCC CCL 10) following the electroporation protocol in the Gibco SFV Gene Expression System manual for producing a recombinant viral stock.
  • BHK, ATCC CCL 10 Baby Hamster Kidney cells
  • the resulting viral stocks were harvested and used to infect various mammalian host cells.
  • Glycosylated proteins are usually retarded to a greater degree in an electrophoretic separation system than is the corresponding unglycosylated protein. Therefore, they appear to have higher molecular weights than the corresponding unglycosylated protein. This was demonstrated for proteins expressed from the pOJ850 vector as follows. Medium in which cells containing the pOJ850 vector had been cultured was incubated with a preparation having N- glycosidase F activity (Endoglycosidase F from Flavobacterium meningospticum, Boehringer Mannheim Biochemica, Indianapolis, IN, Catalog No. 878740) overnight at 37°C ("digested sample”) .
  • N- glycosidase F activity Endoglycosidase F from Flavobacterium meningospticum, Boehringer Mannheim Biochemica, Indianapolis, IN, Catalog No. 878740
  • Stable cells lines capable of expressing glycosylated obesity proteins analogs of the present invention were prepared. Standard techniques described in Current Protocols in Molecular Biology, Volumes 1 and 2, John Wiley and Sons, New York, (1998) were used to: 1) excise from the vectors described above approximately 450 bp fragments containing the coding sequences for obesity protein and obesity protein analogs and purify the same; 2) ligate the fragments into a vector having a selectable marker, an internal ribosomal entry site (IRES) , and the coding sequence for a green fluorescence protein (EGFP) ;
  • IVS internal ribosomal entry site
  • Bam HI was used to digest samples of the pOJ836, pOJ848, and pOJ850 vectors described above, to remove an approximately 450 bp obesity protein fragment.
  • the fragments were purified and then were ligated into a Bel I digested/calf intestinal alkaline phosphatased vector similar to the pIRES-EGFP vector (Clonetech, Palo Alto, CA) , additionally modified to have a suitable selectable marker, such as the SV40-DHFR marker.
  • the vectors were transformed into E. coli DHIOB cells (Gibco BRL) and plated on agar medium plates containing 100 ⁇ g/ml ampicillin. Colonies were selected and grown in 3 mL liquid cultures. Plasmid DNA was isolated. A Pst I diagnostic digest was done to determine which clones contained the proper inserts.
  • pOJ862 was derived from pOJ836, pOJ863 from pOJ848, and pOJ864 from pOJ850.
  • Fsp I was used to linearize the plasmids.
  • Samples of linearized plasmids were spun through an enzyme removal filter, and concentrated on a DNA microconcentrator .
  • the concentrated samples of DNA were transformed into AV12-664 RGT18 cells using Fugene reagent (FuGene 6 Transfection Reagent, Boehringer Mannheim, Indianapolis, IN) .
  • the AV12 cell line (AV12-664, ATCC CRL-9595) was isolated originally from a tumor induced by subcutaneous inoculation of adenovirus 12 (Adl2) into newborn Syrian hamsters. Southern blot analysis of the AV12 cell line suggests that there are two copies of the entire Adl2 genome at two separate integration sites.
  • the AV12 cell line expresses the early region IA and IB proteins of Adl2 (proteins responsible for the induction and maintenance of the transformed phenotype) . Whether other Adl2 proteins are expressed has not been determined.
  • the AV12 line exhibits an epitheloid morphology, although the exact cell type from which it originated cannot be determined.
  • a number of recombinant proteins have been expressed from the GBMT promoter (cDNA promoter in pIGl and pIG3) in AV12 cells [Berg, D. T., et al .
  • the AV12-RGT18 cell line was isolated following transfection of the AV12 line with a glutamate transporter [Desai, M. A., et al . , Molecular Pharmacology 48:648-657 (1995)] .
  • the transfected cells were cultured without selection for 2 days, and were then subjected to selection by adding a medium containing 250 nM methotrexate (MTX) in order to select for cells containing integrated DNA. All subsequent culture, unless otherwise noted, was in medium containing MTX for selection. The cultures were incubated for 3 days under MTX selection, then split 1:5, 1:10, and 1:50 into 150 mm dishes. The dishes were incubated for 23 days, during which time the medium was replaced every 2-3 days. Colonies were trypsinized, the cells concentrated by centrifugation, the pellet resuspended in 10ml of medium, and the cell suspension split into T150 flasks at 1:10.
  • MTX methotrexate
  • the flasks were incubated for 4 days until the 1:10 split was 90-100% confluent.
  • the cells were then sorted by green fluorescence (top 5% brightest green) into pools of 10,000 cells in 20% conditioned media containing 100 nM MTX. After incubation for one day, the medium was replaced with 50% conditioned medium containing no MTX. After another day of incubation, the medium was replaced with 50% conditioned medium containing 250 nM MTX. After a further four days of incubation, the medium was replaced with fresh 50% conditioned medium containing 250 nM MTX. After another three days, the old medium was replaced with fresh medium containing 250 nM MTX, without conditioning. Conditioned medium was not used again until the second sorting.
  • the cultures were split in 6-well dishes (60% confluent) 1:30 into T75 flasks. These were incubated for three days, and then the cultures were split (70-100% confluent) 1:2 and 1:4 into T75 flasks. After one day, the colonies were trypsinized. The 1:2 and 1:4 splits were mixed, and then the cells were sorted by green fluorescence (top 5% brightest green) into pools in a 6-well plate at 10,000 cells into 50% conditioned media with no MTX. The cells were incubated three days, after which the medium was replaced with fresh, unconditioned medium containing 250nM MTX. Conditioned medium was not used again until the third sorting.
  • the cultures were split in 6-well dishes (100% confluent) 1:7.5 into T75 flasks. After this, the pOJ863 and pOJ864 cultures were maintained for about a month, splitting the cultures 1:10 or 1:20 every 3 or 4 days .
  • MTX amplification was initiated at a concentration of 1 ⁇ M MTX.
  • Cells were passed until they grew to confluency within 3 days after a 1:20 split. Then, amplification was increased to 4 ⁇ M MTX using the same process. After the cultures were adapted to 4 ⁇ M MTX, the cells were sorted by green fluorescence for the third time and passaged continually thereafter.
  • the pOJ864 clone which was prepared as described above, was grown in DMEM-F12 3:1 (Gibco-BRL, Grand Island,
  • MTX MTX
  • the supernatant was harvested 24 hours later, and replaced with fresh serum-free medium. A second collection was harvested 24 hours later.
  • the supernatants were pooled (5 liters) and concentrated by microfiltration using a 10 kDa microfilter (YM10, Amicon, Beverly, MA) prior to purification of the glycosylated obesity protein analog.
  • the glycosylated obesity protein analog was purified in three steps, using first a lectin binding column, then a nickel affinity column, and finally gel chromatography column. Concentrated supernatant was allowed to bind to a concanavalin A-sepharose resin in 20 mM Tris, 0.5 M NaCl, pH 7.4 (binding buffer) by incubation overnight. The slurry was poured into a column and after the resin packed, the bed was washed with ten column volumes of binding buffer. The glycosylated protein was eluted with ten column volumes of 0.5 M methyl- ⁇ -D mannopyranoside (Sigma Chemical Company, St. Louis, MO).
  • Fractions containing the glycosylated obesity protein analog were carried to the next step .
  • the eluted protein from the above step was further purified on a iminodiacetic acid sepharose 6B column (Sigma Chemical Company) .
  • the selected fractions from the Con A column were incubated with the iminodiacetic acid resin overnight.
  • the suspension was poured into a column, and after the resin packed, the column was washed with ten column volumes of a solution containing 6.5 mM sodium hydrogen phosphate, 2.7 mM potassium chloride, 137 mM sodium chloride, 1.5 mM potassium dihydrogen phosphate, pH 7.5 (wash buffer) .
  • the protein was eluted using a 2-step pH gradient.
  • glycosylated obesity protein analog Fractions containing glycosylated obesity protein analog were verified using SDS-PAGE with 12% gels. Fractions were pooled on the basis of the electrophoresis results. N-terminal amino acid sequencing analysis for 8-10 cycles showed identity with the expected sequence of the obesity protein analog. The yield of glycosylated obesity protein analog after expression and purification was 0.5 mg/L/10 9 cells. Protein was quantitated using the BCA protein assay kit (Pierce Biotech, Rockford, IL) .
  • Example 1 In Vivo Biological Testing Parabiotic experiments suggest that obesity protein is released by peripheral adipose tissue and that the protein is able to control body weight gain in normal, as well as obese mice. Experiments are done with five to six month old male, inbred normal ICR mice, inbred normal (OB/?) , obese-diabetic mice (ob/ob) from the Jackson Laboratories (Bar Harbor, Maine) or Harlan (England) mice. Both normal and diabetic mice are housed three or six per plastic cage (with bedding) and water and feed are available- ad liJbi um. The temperature of animal rooms is maintained at 23 ⁇ 2°C and lights are on from 0600 to 1800 h. Blood samples are collected from the tail vein.
  • a formulation of a test protein is administered by any one of several routes (e.g., i.v., s.c, i.p., or by minipump or cannula) .
  • Food and water consumption, body weight gain, plasma chemistry or hormones are monitored over various time periods .
  • Suitable test animals include normal mice (ICR, etc.) and obese mice
  • test protein may be administered to animals that are thought to lack the receptor (fa/fa or cp/cp rats) .
  • 50 ⁇ L of a formulation containing 30 ⁇ g of a protein of the present invention together with glycerin (16 mg/mL) and phenol (2 mg/mL) is injected subcutaneously into ob/ob mice in a single injection per day for four days.
  • the average daily food intake and cumulative change in body weight from time zero for the ob/ob mice are measured.
  • the present proteins will affect feed intake and body weight in a manner consistent with the demonstrated effect of obesity protein. Proteins demonstrating activity in these models will demonstrate similar activity in other mammals, particularly humans.
  • a suitable model is to inject the test protein directly into the brain via the lateral or third ventricles (e . g. , i.c.v.), or directly into specific hypothalamic nuclei (e . g. , arcuate, paraventricular, perifornical nuclei) .
  • the same parameters as above could be measured.
  • the release of neurotransmitters that are know to regulate feeding or metabolism such as, neuropeptide Y (NPY) , galanin, norepinephrine, dopamine, or ⁇ -endorphin, could be monitored.
  • a glycosylated obesity protein analog of the present invention is administered intravascularly (i.v.) to a group of animals.
  • An obesity protein, or an analog thereof is administered to a different group of animals, which serves as the control group.
  • blood samples are removed from the animals in each group, and the concentration of glycosylated obesity protein analog, or the control protein, is assayed.
  • the assay may be, for example, an ELISA or an RIA [Ma, Z., et al . , Clin . Chem. 42:942-946 (1996); Human Leptin Assay Kit, LINCO Research, Inc., St. Charles, MO.].
  • glycosylated obesity protein analogs of the present invention will be found to persist in the blood for a longer period than non-glycosylated obesity protein analogs.
  • the physical and chemical stability of the present compounds is demonstrated as follows.
  • the starting solutions contain purified obesity protein in phosphate- buffered saline (Gibco BRL, Dulbecco ⁇ s PBS without calcium phosphate or magnesium phosphate, from Life Technologies, Inc., Grand Island, NY) .
  • the protein concentrations are generally determined by their absorbance at 280 nm.
  • the obesity protein solutions are then added to 2- mL glass autosampler vials (Varian Instrument Group, Sunnyvale, CA) each containing 15 Teflon balls one-eighth inch in diameter (Curtin Matheson Scientific, Florence, KY) . Air bubbles are removed from the solutions in the vials with gentle shaking. The vials are slightly overfilled at the top and then closed with the Teflon-coated seal and screw cap. A separate vial is used for each shake test time period that is to be evaluated. The test vials are placed in a rotation device in an incubator set precisely at 40°C. The vials are rotated end-over-end at a rate of 30 revolutions per minute, allowing the Teflon beads to move gently from the top of the vial to the bottom while remaining completely in the solution.
  • the test vials are placed in a rotation device in an incubator set precisely at 40°C. The vials are rotated end-over-end at a rate of 30 revolutions per minute, allowing the Tefl
  • the contents of the vials are removed and centrifuged 5 minutes at ambient temperature (Fisher Scientific Model 235C Centrifuge) .
  • the protein concentrations in the clear supernatants are determined again by the UV absorbance or SEC techniques.
  • the percent of obesity protein remaining in solution is calculated from the Ob concentrations in the pH-adjusted starting solutions and in the supernatants after the shake test .
  • the physical stability is alternatively demonstrated by storing formulations of the proteins of the invention at various temperatures and time periods, and measuring their turbidity by nephelometry, using, for example a Hach Ratio Turbidimeter (Model 18900, or 2100N for values above 200) calibrated with Hach Formazin Turbidity Standard.
  • a formulation containing un-glycosylated human obesity protein is run as a control .
  • the physical stability is also assessed by incubating soluble formulations of the present proteins for various periods of time, and assaying the amount of soluble protein remaining after various periods of time. For example, the concentration of protein remaining in solution after 3 days at 4°C and 37°C is a measure of the physical stability of the protein formulations. Size-exclusion chromatography may be used to assess the concentration of protein remaining in solution. Samples are first centrifuged, and then the supernatants are injected onto a column of size exclusion resin, such as, an analytical Superdex-75 (Pharmacia) column, equilibrated with a suitable buffer, such as, PBS (Dulbecco's Phosphate-Buffered Saline, Gibco BRL) .
  • a suitable buffer such as, PBS (Dulbecco's Phosphate-Buffered Saline, Gibco BRL) .
  • the column is eluted with the same buffer solution, and concentration of the protein in the eluant is monitored at 214 nm.
  • concentration of the protein in the eluant is monitored at 214 nm.
  • the area under the absorbance peak for the obesity protein sample is integrated and compared with the area under a peak of a standard, such as, native obesity protein, and also against a control, such as, native obesity protein that is incubated under the same conditions as the sample protein.
  • Physical instability of a formulation of a protein may be indicated by an increase in the hydrodynamic radius of particles in the formulation over time . Such an increase in particle size is usually caused by aggregation of proteins in the formulation. Therefore, an alternate method for assessing physical stability is dynamic light scattering (DLS) , using, for example, a laser light scattering instrument ( e . g. , Model BI9000, Brookhaven Instruments, Holtsville, NY) . Using the measurements obtained with this instrument, and with reasonable assumptions about the viscosity and diffusional coefficient, this method permits calculation of the hydrodynamic radius of the solute through the Stokes-Einstein equation [Koppel, D. E., J " . Chem . Phys .
  • glycosylated obesity protein analogs of the present invention aggregate less rapidly than obesity protein analogs that are not glycosylated.
  • the compounds are active in at least one of the above biological tests and are anti-obesity agents. As such, they are useful in treating obesity and those disorders implicated by obesity.
  • the proteins are not only useful as therapeutic agents.
  • One skilled in the art recognizes that the proteins are useful in the production of antibodies for diagnostic use and, as proteins, are useful as feed additives for animals.
  • the compounds are useful for controlling weight for cosmetic purposes in mammals. A cosmetic purpose seeks to control the weight of a mammal to improve bodily appearance. The mammal is not necessarily obese. Such cosmetic use forms part of the present invention.

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Abstract

La présente invention se rapporte à des analogues de la protéine de l'obésité glycosylée possédant des sites consensus de glycosylation à liaison N. D'une manière plus générale, l'invention se rapporte à la protéine de l'obésité glycosylée et à des analogues de ladite protéine, qui possèdent une meilleure stabilité physique ou une demi-vie plasmatique plus longue, ou encore les deux, en comparaison à la protéine de l'obésité non glycosylée. On effectue la glycosylation par l'adjonction d'un fragment de glycosyle à un atome d'azote de la protéine de l'obésité ou d'un analogue de la protéine de l'obésité. L'invention concerne également des composés d'ADN comprenant des analogues de la protéine de l'obésité, des cellules hôtes transformées, des préparations à base de protéine de l'obésité glycosylée et de ses analogues, des procédés pour traiter l'obésité et un procédé pour créer des sites de glycosilation dans la protéine de l'obésité ou dans ses analogues.
PCT/US1998/023323 1997-10-31 1998-11-02 Analogues de la proteine de l'obesite glycosylee WO1999023115A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000047741A1 (fr) * 1999-02-12 2000-08-17 Amgen Inc. Compositions glycosylees de leptine et procedes correspondants
WO2008087121A1 (fr) * 2007-01-17 2008-07-24 Merz Pharma Gmbh & Co. Kgaa Utilisation d'un surfactant dans la préparation d'une formulation destinée au traitement de maladies adipeuses

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JPH05178899A (ja) * 1990-07-31 1993-07-20 Toray Ind Inc 修飾ポリペプチドおよびそれを有効成分とする血小板減少症治療剤
US5612029A (en) * 1992-06-03 1997-03-18 Genentech, Inc. Tissue plasminogen activator glycosylation variants with improved therapeutic properties
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DATABASE BIOSIS ON STN, WOLFF et al., "Release and Preparation of Intact and Unreduced N-Linked Oligosaccharides from SF-9 Insect Cells"; & PREP. BIOCHEM. BIOTECHNOL., 1999, Vol. 29, No. 1, pages 1-21. *
HALTIWANGER et al., "Breakthroughs and Views: O-Glycosylation on Nuclear and Cytoplasmic Protein: Regulation Analogous to Phosphorylation?", BIOCHEM. BIOPHYS. RES. COMM., 1997, Vol. 231, pages 237-242. *
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000047741A1 (fr) * 1999-02-12 2000-08-17 Amgen Inc. Compositions glycosylees de leptine et procedes correspondants
WO2008087121A1 (fr) * 2007-01-17 2008-07-24 Merz Pharma Gmbh & Co. Kgaa Utilisation d'un surfactant dans la préparation d'une formulation destinée au traitement de maladies adipeuses

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