WO1996031526A1 - Agents antiobesite - Google Patents

Agents antiobesite Download PDF

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Publication number
WO1996031526A1
WO1996031526A1 PCT/US1996/004909 US9604909W WO9631526A1 WO 1996031526 A1 WO1996031526 A1 WO 1996031526A1 US 9604909 W US9604909 W US 9604909W WO 9631526 A1 WO9631526 A1 WO 9631526A1
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WO
WIPO (PCT)
Prior art keywords
dimer
fusion protein
protein
monomer
antibody
Prior art date
Application number
PCT/US1996/004909
Other languages
English (en)
Inventor
Nigel Beeley
Timothy J. Rink
Keith Alan Albrandt
Michael Edward Sierzega
Susan M. Janes
Kathryn S. Prickett
Julie L. Phelps
Mark Chun
Douglas M. Park
Daniel E. Beidler
Original Assignee
Amylin Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amylin Pharmaceuticals, Inc. filed Critical Amylin Pharmaceuticals, Inc.
Priority to AU55395/96A priority Critical patent/AU5539596A/en
Publication of WO1996031526A1 publication Critical patent/WO1996031526A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • kilobase (kb) adipose tissue messenger RNA with a highly conserved 167-amino-
  • Zhang et al. at 429. Alignment of the predicted human and mouse ob amino-acid sequences reported in Zhang et al. is set forth below:
  • obesity is currently a poorly treatable, chronic, essentially intractable metabolic disorder. Not only is obesity itself undesirable for social reasons, but obesity also carries serious risk of co- morbidities including, Type 2 diabetes, hypertension, atherosclerosis, degenerative reasons
  • Type 2 diabetes in which dieting and weight loss are the primary
  • the present invention is directed to the manufacture and use of dimeric
  • the invention relates to ob dimers, which include human
  • the invention also relates to ob dimer fusion proteins, including ob dimer fusion
  • poly-histidine and the eight amino acid marker peptide known in the art as
  • pure is meant purity greater than or equal to about 90% and particularly greater
  • tissue total RNA preferably adipose tissue total RNA.
  • tissue poly-A + RNA is used in place of tissue total RNA or adipose tissue total
  • monomer may optionally be converted to ob dimer fusion protein as described
  • Preferred purification methods include the use of an
  • osmotic shock protocol which incorporates one or more specific protease inhibitors, preferably Peflabloc SC, followed by the addition of BisTris-propane, or buffers of a
  • the invention provides for the chemical synthesis
  • dimerization may be achieved by
  • the invention provides ob dimer and ob
  • Such compounds and compositions including obesity and diabetes, particularly Type 1,
  • dimer fusion proteins include human ob dimers and human ob dimer fusion proteins
  • rat ob dimers and rat ob dimer fusion proteins mouse ob dimers and mouse ob dimer fusion proteins, as well as other vertebrate ob dimers and vertebrate ob dimer fusion
  • Ob monomer proteins and ob monomer fusion proteins include human ob
  • ob dimer a vertebrate ob monomer and ob monomer fusion proteins.
  • these ob dimer preferably, these ob dimer
  • ob monomer compounds are prepared in a stable lyophilized form as trifluoroacetate, acetate, hydrochloride, or ammonium bicarbonate salts, most
  • ob dimer and ob monomer compounds preferably ammonium bicarbonate salts.
  • Preferred stable solutions of these ob dimer and ob monomer compounds are prepared using BisTris-propane, or buffers of
  • Treatment methods comprise the administration of a therapeutically effective amount of a pharmaceutical composition comprising an ob dimer and/or ob
  • compositions may be administered separately or together with other compounds and
  • compositions that exhibit a short-term satiety action including but not limited to other compounds and compositions that comprise an amylin or an amylin agonist.
  • Suitable amylins include, for example, human amylin and rat amylin.
  • Suitable amylin agonists include, for example, [Pro ⁇ - ⁇ 'J-human amylin and salmon
  • the present invention provides novel antibodies,
  • polyclonal antibodies preferably monoclonal antibodies, and antibody fragments which can be produced in mice or by recombinant cell lines or by hybrid
  • the antibodies being characterized in that they have certain predetermined
  • antibodies and antibody fragments are useful in methods for the purification of ob
  • acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E),
  • glycine Gly or G
  • histidine His or H
  • isoleucine lie or I
  • leucine Leu or L
  • lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or
  • peptide refers to a sequence of amino acids linked predominantly
  • protein refers to a molecule comprised of one or more peptides.
  • cDNA refers to complementary deoxyribonucleic acid.
  • nucleic acid refers to polymers in which bases (e.g., purines or
  • Nucleic acids include
  • mRNA refers to messenger ribonucleic acid. 6/31526 PC17US96/04909
  • nucleic acid sequence refers to the sequence of nucleosides
  • recombinant refers to a DNA molecule comprising pieces of DNA that are not normally contiguous, or to a protein expressed therefrom.
  • FIGURE 1 shows the nucleotide and deduced amino acid sequences of the
  • FIGURE 2 shows the change in food intake in ob/ob mice administered
  • FIGURE 3 shows the change in food intake in ob/ob mice administered
  • FIGURE 4 shows the change in food intake in ob/ob mice administered
  • FIGURE 5 shows the change in food intake in ob/ob mice administered
  • FIGURE 6 shows the change in food intake in ob/ob mice administered
  • FIGURE 7 shows the change in food intake in NIH/Sw mice administered
  • FIGURE 8 shows the change in body weight in ob/ob mice administered
  • FIGURE 9 shows the change in body weight in ob/ob mice administered various doses of rat ob protein and two doses of Met-rat ob protein.
  • FIGURE 10 shows the change in body weight in ob/ob mice administered
  • FIGURE 11 shows the change in body weight in ob/ob mice administered various doses of rat ob protein and two doses of FLAG-rat ob protein.
  • FIGURE 12 shows the change in body weight in ob/ob mice administered
  • FIGURE 13 shows the change in body weight in NIH/Sw mice administered
  • the present invention is directed to dimeric forms of the ob gene product, including ob dimers and ob dimer fusion proteins, as well as to ob monomer fusion
  • compositions including but not limited to those subjects who would benefit from
  • Ob dimers include dimers of the ob gene product from any vertebrate source
  • rat ob gene sequence useful in preparing an ob dimer or other ob product is described herein and
  • GTT as the codon for Val 22 (in place of the naturally-occurring GTG codon)
  • CCG as the codon for Pro 23 (in place of the
  • GTT as the codon for Val 22 (in place of the naturally-occurring GTG codon)
  • CCG as the codon for Pro 23 (in place of the
  • the human ob DNA sequence also contains the amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino acids, amino
  • An especially preferred mouse ob DNA sequence includes GTT as the codon for Val 22 (in place of the naturally-occurring GTG codon) and CCG as the codon for
  • ob dimers and ob dimer fusion proteins of the present invention include those having variations in a known or disclosed ob gene sequence or sequences, including fragments, naturally occurring mutations, allelic variants, randomly generated
  • DNA to be incorporated into a cell that will allow the production of the protein for which the original DNA encodes.
  • tissue mRNA tissue mRNA
  • tissue total RNA preferably adipose tissue total RNA
  • genetic information include isolating mRNA from an organism, converting it to its
  • mRNA is first reverse-transcribed to form a single-stranded cDNA
  • RNA-directed DNA polymerase e.g., reverse transcriptase
  • double-stranded cDNA is produced and inserted into cloning or expression vectors by any one of many known techniques, which depend
  • Expression vectors refer to vectors which are
  • One method of preparing the products of the invention includes the steps of
  • the products of the invention may also be prepared by methods that do not
  • Examples 1 and 2 describe methods used to isolate
  • ob dimers, ob dimer fusion proteins and ob monomer fusion proteins of the invention can be
  • RNA extraction including the acid guanidinium thiocyanate
  • Example 2 details the extraction of rat adipose tissue total RNA and the preparation of oligonucleotide primers for use in the isolation and
  • RNase ribonuclease
  • preparing cDNA include those described in Example 2 wherein total RNA isolated
  • Cloning vectors include a DNA sequence which accommodates the cDNA.
  • the vectors containing the amplified cDNA or cDNA library are introduced into host cells that can exist in a stable manner and provide an environment in which the
  • cloning vector is replicated.
  • Suitable cloning vectors include plasmids,
  • Preferred cloning vectors include plasmids.
  • Isolated plasmids, DNA sequences or synthesized oligonucleotides are cleaved, tailored and religated in the form desired.
  • site-specific cleavage of cDNA is performed by treating with suitable restriction enzyme under conditions which are generally understood in
  • Cloning vectors containing the desired cDNA are introduced into host cells
  • Cloning vectors containing a cDNA library prepared as disclosed are
  • Preferred host cells include bacteria
  • Hybridization probes and primers are oligonucleotide sequences which are
  • diethylphosphoramidites are used as starting materials and may be synthesized as
  • Probes differ from primers in that they are labeled with an enzyme, such as horseradish peroxidase, or with a radioactive atom, such as 32 P,
  • a synthesized probe is radio-labeled by nick translation
  • T4 bacteriophage polynucleotide kinase T4 bacteriophage polynucleotide kinase.
  • Useful hybridization probes and amplification primers include
  • oligonucleotide sequences which are complementary to a stretch of the cDNA
  • hybridization probes are oligonucleotide sequences encoding substantially all of the amino acid sequence of rat, mouse, or human ob protein. Other appropriate probes
  • oligonucleotide sequences are Especially preferred as amplification primers.
  • a preferred cDNA molecule encoding a vertebrate protein of the present invention can be identified by screening or amplification methods through its ability to hybridize to these probes or
  • amplification include the use of the polymerase chain reaction (PCR). See, e.g.,
  • PCR is an in vitro amplification method for the synthesis of specific DNA sequences.
  • two oligonucleotide primers that hybridize to opposite strands
  • polymerase results in numbers of copies of cDNA, whose termini are defined by the
  • restriction sites such as restriction sites or translational signals (signal sequences, start and/or stop
  • a recombinant cDNA molecule of the present invention is incorporated into an expression vector, this expression vector is introduced into an appropriate
  • the host cell is cultured, and the expressed protein is isolated.
  • Expression vectors are DNA sequences that are required for the transcription
  • vectors can express either procaryotic or eucaryotic genes in a variety of cells such as bacteria, yeast, mammalian, plant and insect cells. Proteins may also be expressed in a number of virus systems.
  • Suitably constructed expression vectors contain an origin of replication for autonomous replication in host cells, or are capable of integrating into the host cell
  • Such vectors will also contain selective markers, a limited number of
  • Promoters are DNA sequences that direct RNA polymerase to bind to DNA and
  • RNA synthesis initiates RNA synthesis; strong promoters cause such initiation at high frequency.
  • the preferred expression vectors of the present invention are operatively linked to a
  • cDNA or recombinant cDNA of the present invention i.e., the vectors are capable of directing both replication of the attached cDNA or recombinant cDNA molecule and
  • Expression vectors may include, but are not limited to cloning vectors, modified
  • cloning vectors and specifically designed plasmids or viruses. With each type of host cell certain expression vectors are preferred, as described below. Procaryotes may be used and are presently preferred for expression of the ob
  • Suitable bacteria host cells include the various strains of E. coli, Bacillus
  • Suitable vectors for E. coli are derivatives of
  • pBR322 a plasmid derived from and E. coli species by Bolivar et al.. Gene, 2: 95 (1977).
  • Common procaryotic control sequences which are defined herein to include
  • promoters for transcription for transcription, initiation, optionally with an operator, along with
  • ribosome binding site sequences include the beta-lactamase and lactose promoter (Chang et al.. Nature, 198: 1056 (1977)), the tryptophan promoter system (Goeddel et al.. Nucleic Acids Res., 8: 4057 (1980)) and the lambda-derived ? promoter and
  • Preferred procaryote expression systems include E. coli and their expression vectors
  • E. coli strains W3110 and JM105 such as E. coli strains W3110 and JM105, with suitable vectors, as described in
  • Example 5 Especially preferred is the use of E. coli strain BL21(DE3), with
  • Eucaryotes may be used for expression of the proteins of the present
  • Eucaryotes are usually represented by the yeast and mammalian cells. Suitable yeast host cells include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable mammalian host cells include COS and CHO (Chinese Hamster Ovary) cells.
  • Expression vectors for the eucaryotes are comprised of promoters derived
  • yeast cell expression vectors include promoters for synthesis of glycolytic enzymes, including those for the 3-phosphoglycerate kinase gene in Saccharomyces cerevisiae (Hitzman et al.. J.
  • Suitable promoters for mammalian cell expression vectors include the early
  • promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or avian sarcoma viruses. Suitable viral and mammalian enhancers may also be used.
  • Suitable promoters for plant cell expression vectors include the nopaline
  • Suitable promoters for insect cell expression vectors include modified versions of
  • vector comprises a baculovirus polyhedrin promoter under whose control a cDNA
  • molecule encoding a protein can be placed.
  • Another method of producing an ob dimer comprises the steps of culturing a
  • a human ob protein under conditions that favor the production of said vertebrate ob protein as a dimer, and isolating the ob dimer expressed by the
  • Still another method of producing a recombinant ob dimer comprises the steps of culturing a transformed host cell containing a DNA sequence encoding a
  • vertebrate ob protein preferably a human ob protein, under conditions that favor the
  • Dimerization may be achieved by initially treating ob monomer protein with
  • a reducing agent such as mercaptoethanol or dithiothreitol in an appropriate buffer at
  • oxidation typically used are O 2 /copper, mercury salts, etc.
  • Folding aids can also be
  • proteins such as albumins, chaperones, monoclonal antibodies,
  • Ob dimer fusion proteins may be produced using similar methods.
  • steps of constructing a vertebrate cDNA library preferably a vertebrate adipose
  • cDNA library and more preferably a human adipose cDNA library; ligating the cDNA library into a cloning vector; introducing the cloning vector containing the
  • RNA adipose tissue total RNA
  • a preferred peptide for preparation of an ob dimer fusion peptide is the
  • FLAG is an octapeptide with the amino acid sequence DYKDDDDK.
  • Antibodies are available which specifically recognize this sequence, thus allowing
  • reporter peptide allows identification, purification and liberation of a given protein to which it is attached. Additionally, cloning into the pFLAG-ATS vector
  • Another method of producing an ob dimer fusion protein comprises the steps
  • vertebrate ob protein preferably a human ob protein, coupled to a marker or other
  • Still another method of producing an ob dimer fusion protein comprises the steps of culturing a transformed host cell containing a DNA
  • vertebrate ob fusion protein as a monomer, isolating the ob fusion protein expressed
  • E. coli BL21(DE3) cells which are grown at about 25° to about 30°C in media containing a supplemental carbon source, preferably glucose, for enhanced expression.
  • a supplemental carbon source preferably glucose
  • Intracellular expression can be used to make proteins in E. coli, but the
  • a chaotropic agent such as urea in ammonium bicarbonate buffer
  • the protein using a cellulose-based anion exchange chromatography resin, preferably
  • the invention also provides for the preparation of ob monomer protein
  • phase peptide synthesis and solution chemistries offers a further method of
  • Solid phase synthesis is commenced from the C-terminus of the peptide by coupling a protected amino acid or peptide to a suitable insoluble resin. Suitable
  • resins include those containing chloromethyl, bromomethyl, hydroxymethyl,
  • amino acid can be directly coupled.
  • this solid phase In one embodiment of this solid phase
  • the synthesis may be done manually, by using
  • amino group of the resin-bound amino acid can be carried out according to
  • BOP benzotriazole-1-yl-oxy-tris (diamino) phosphonium hexafluorophosphate
  • Woodward reagent K method Woodward reagent K
  • the completed peptide may be cleaved from the resin by treatment with
  • the completed peptide may be
  • the cleaved peptide is dissolved in water or
  • the purified peptide is then allowed to refold and establish proper disulfide bond formation by dilution to an appropriate
  • peptide concentration for example from about 0.025 mM to about 0.25 mM, in an
  • disulfide bond formation may be performed by using a
  • cellulose anion exchange chromatography preferably employing a DE-52 resin.
  • fractions containing the purified peptide Upon collection of fractions containing the purified peptide, the fractions are pooled
  • the rat ob protein may be prepared in this way.
  • the rat ob protein may be prepared in this way. In the absence of signal sequence, the rat ob protein
  • Fragment 3 requires resin cleavage conditions which place a carboxylic acid
  • Fragments 1 and 2 require their N-terminal protecting groups
  • Fragment 3 also requires subsequent N-terminal deprotection while leaving side chain and C-terminal protecting groups intact.
  • Fragment 3 for between 3 and 48 hours at room temperature.
  • Fragment 3 for between 3 and 48 hours at room temperature.
  • N-(3-Dime ylam opropyl)-N'-ethylcarbodiimide can be used in place of DCC.
  • Fragment 1 is then activated, typically with DCC/N-hydroxybenzotriazole in a suitable solvent, typically DMF.
  • a suitable solvent typically DMF.
  • the activated Fragment 1 is then reacted with C- 6/31526 PO7US96/04909
  • cysteines has been incorporated into the synthesis, by a sequence of selective
  • the ob proteins of the present invention may be isolated, for example from
  • ob dimer fusion protein include chromatography of an extract through
  • a cellulose-based cation exchange resin preferably CM-52.
  • any such chromatography procedures may be selected by methods such as gel chromatography
  • mice feeding in mice, as described in Example 23 below, or by a combination of such
  • fusion protein such as a FLAG-ob monomer fusion protein or a FLAG-ob dimer
  • fusion protein includes chromatography using an anti-FLAG affinity gel, provided by the manufacturer as a suspension of agarose beads covalently linked to anti- FLAG monoclonal antibodies, optionally followed by anion and/or cation exchange
  • FLAG-ob protein on the FLAG Ml affinity resin could be limited by competition for binding sites between intact FLAG-ob protein and an N-terminal fragment of the
  • Anion exchange fractionation may first be used to remove any
  • ob dimer or ob dimer fusion protein illustrated by purification of FLAG ob
  • Example 6 describes the purification of a FLAG-ob protein preparation
  • fusion protein and ob dimer fusion protein but predominantly monomer, isolation and purification of ob dimer fusion protein to a purity of about 90% can be
  • the present invention also contemplates antibodies and immunoassays useful
  • an ob protein or protein fragment for example an ob dimer and or ob dimer fusion protein, and antibodies useful therein.
  • Patent 4,376,110 entitled “Immunometric Assays Using Monoclonal Antibodies
  • test may include a known ob dimer and/or ob dimer fusion protein positive, control and a known non- ob dimer and/or non-ob dimer fusion protein negative control.
  • control may include a known ob dimer and/or ob dimer fusion protein positive, control and a known non- ob dimer and/or non-ob dimer fusion protein negative control.
  • Other control may include a known ob dimer and/or ob dimer fusion protein positive, control and a known non- ob dimer and/or non-ob dimer fusion protein negative control.
  • samples may include an ob monomer or ob monomer fusion protein.
  • the immunoassay is a sandwich immunoassay, and comprises the
  • anti-ob dimer fusion protein antibody preferably a monoclonal antibody
  • reaction of the immobilized antibody and labeled antibody with the sample may be carried out either simultaneously or separately.
  • one of the antibody pair used in such an assay preferably the labeled
  • antibody is an anti-ob monomer antibody or an anti-ob monomer fusion protein
  • bound antibody may be the same or different anti-ob monomer antibodies.
  • Suitable antibodies for example anti-ob dimer and/or anti-ob dimer fusion
  • inventions are specific to an ob dimer and/or ob dimer fusion protein over an ob
  • Such antibodies as well as suitable anti-ob monomer antibodies or anti-ob monomer fusion protein antibodies, can be
  • the immunogen may be either crude or partially purified, and is administered to a mammal, such as mice, rats or rabbits,
  • myeloma cells having a suitable marker such as 8-azaguanine resistance can be used
  • hybridomas as parent cells which are then fused with the antibody-producing spleen cells to prepare hybridomas.
  • media such as Eagle's MEM, Dulbecco's modified
  • Myeloma parent cells and spleen cells can be suitably fused at a ratio of approximately 1 :4.
  • Polyethylene glycol (PEG) can be used as a
  • Suitable fusing agent typically at a concentration of about 35% for efficient fusion.
  • Resulting cells may be selected by the HAT method. Littlefield, J. W., (1964)
  • hybridomas to identify a clone of hybridoma producing the objective immunoglobulin.
  • the obtained antibody-producing hybridoma can then be cloned
  • FCS fetal calf serum
  • the hybridoma is cultured in vivo, the hybridoma may be implanted in the abdominal
  • Antibodies or the desired binding portions thereof including F(ab) and Fv
  • fragments, along with antibody-based constructs such as single chain Fv's can also be generated using processes which involve cloning an immunoglobulin gene library
  • a vector system is constructed following a PCR amplification
  • a sandwich immunoassay for ob dimers and/or ob dimer fusion proteins can
  • Antibodies according to the present invention can suitably be immobilized
  • ob dimer, anti-ob monomer, anti-ob monomer fusion protein, and/or anti-ob dimer fusion protein monoclonal antibody can be stored cold in the presence of
  • Labeled anti-ob dimer, anti-ob monomer, anti-ob monomer fusion protein, and anti-ob dimer fusion protein antibodies in accordance with the present invention can suitably be prepared by labeling anti-ob dimer, anti-ob monomer, anti-ob
  • monomer fusion protein and anti-ob dimer fusion protein antibodies with any substance commonly used for an immunoassay including radioisotopes, enzymes,
  • Radioisotopes and enzymes are preferably used.
  • the antibody is preferably labeled with ,25 I using
  • the antibody is labeled with an enzyme such as horseradish peroxidase, ⁇ -D-
  • galactosidase or alkaline phosphatase by conventional methods including the
  • the activity of the label can be detected by conventional methods. If
  • radioisotopes are used as labels
  • the activity of the label can be detected using an appropriate instrument such as a scintillation counter. If enzymes are used as labels,
  • the activity of the label can be detected by measuring absorbance, fluorescence
  • the present invention also provides a kit for assaying the amount of ob dimer
  • ob dimer fusion protein present in a sample, including for example both
  • kit of the present invention comprises an immobilized anti-ob dimer and or anti-ob dimer fusion protein monoclonal antibody and a labeled anti-ob dimer and/or anti-ob dimer fusion protein monoclonal antibody.
  • anti-ob monomer antibodies and anti-ob monomer fusion protein antibodies can also be used as the immobilized or labeled
  • proteins or both can be measured by this invention.
  • ob dimers ob dimer fusion proteins
  • ob dimer fusion proteins ob monomer fusion proteins
  • Such conditions or disorders include obesity and diabetes, particularly Type 2 diabetes.
  • the compounds of the invention possess activity as
  • anti-obesity agents as evidenced by the ability to reduce feeding in mammals.
  • mice in fasted (and presumably hungry) mice for up to 12 hours following a single injection, with no apparent "catch-up" in food intake over 24 hours, and with no
  • ob dimer fusion proteins and ob monomer fusion proteins thus may also be used to
  • compositions include therapeutically effective agents.
  • Such pharmaceutical compositions include therapeutically effective agents.
  • ob dimer or ob dimer fusion protein or ob monomer fusion protein in pharmaceutically acceptable carriers.
  • therapeutically effective amount is
  • Ob dimers and ob dimer fusion proteins include human ob
  • proteins include human ob monomer fusion proteins, rat ob monomer fusion
  • mouse ob monomer fusion proteins as well as other vertebrate ob
  • Treatment methods comprise the administration of a therapeutically effective amount of a pharmaceutical composition comprising an ob
  • compositions may be administered separately or
  • compositions that comprise an amylin or an amylin agonist. See Lutz et al.. (1994)
  • amylins include, for example,
  • amylin and rat amylin include, for example, 25 ' 28, 29 Pro-human amylin, amylin agonists as described in "Amylin Agonist Peptides and
  • Such salts include but are not
  • salts prepared with organic and inorganic acids for example, HC1, HBr,
  • H 2 SO 4 , H 3 PO 4 trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, toluenesulfonic acid, maleic acid, fumaric acid and camphorsulfonic acid.
  • Salts prepared with bases include ammonium salts, alkali metal salts, e.g., sodium and
  • potassium salts and alkali earth salts, e.g,. calcium and magnesium salts.
  • alkali earth salts e.g,. calcium and magnesium salts.
  • ammonium bicarbonate salts are especially preferred, and are also preferred for preparation of ob
  • the salts may be formed by conventional means, and by reacting
  • solvent such as water which is then removed in vacuo or by freeze-drying or by
  • compositions or products of the invention may conveniently be provided in
  • formulations suitable for parenteral including intravenous,
  • fusion protein of the invention and another shorter-acting satiety agent, such as an
  • amylin or an amylin agonist in a single composition or solution for administration
  • ob monomer protein an amylin or an amylin agonist
  • administration format may best be determined by a medical practitioner for each patient individually.
  • Ob dimers and ob fusion proteins should preferably be formulated in
  • Ob dimers and ob dimer fusion proteins may not be stable or may be partially or wholly denatured at acid pH and
  • the desired isotonicity may be accomplished using
  • dimer or ob fusion protein or ob monomer or ob monomer fusion protein are dimer or ob fusion protein or ob monomer or ob monomer fusion protein.
  • the proteins of the invention are prepared as ammonium bicarbonate salts.
  • the proteins of the invention are prepared as ammonium bicarbonate salts.
  • the proteins of the invention are prepared in BisTris-propane buffer, or buffers of similar structure, with or without a detergent, such as Tween 80, to have a
  • Lyophilized and liquid formulations of ob monomer proteins may also be formulated as described herein and such formulations form a part of the invention.
  • a form of repository or "depot” slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the
  • solutions of the above compositions may be prepared in

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
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  • Gastroenterology & Hepatology (AREA)
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  • Obesity (AREA)
  • Child & Adolescent Psychology (AREA)
  • Immunology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur des protéines incluant diverses formes dimères et monomères d'un produit génique ob, ou des séquences d'ADN codant des protéines ob modifiées, présentant une activité anorexigène et sur leurs procédés de préparation, de purification, de formulation ou d'utilisation, par exemple pour le traitement de différents troubles de la nutrition ou du métabolisme dont l'obésité et le diabète. L'invention porte également sur des anticorps et des dosages immunologiques servant à la détection et à la quantification desdites protéines.
PCT/US1996/004909 1995-04-06 1996-04-05 Agents antiobesite WO1996031526A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU55395/96A AU5539596A (en) 1995-04-06 1996-04-05 Anti-obesity agents

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WO1998028335A1 (fr) * 1996-12-20 1998-07-02 Eli Lilly And Company Proteines anti-obesite
WO1998028414A1 (fr) * 1996-12-20 1998-07-02 Eli Lilly And Company Proteines anti-obesite
WO1998041222A1 (fr) * 1997-03-20 1998-09-24 Eli Lilly And Company Formulations de proteine de l'obesite
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EP0971742A1 (fr) * 1997-01-17 2000-01-19 Eli Lilly And Company Formulations renfermant une proteine de l'obesite
US6025325A (en) * 1995-05-05 2000-02-15 Hoffman-La Roche Inc. Pegylated obese (ob) protein compositions
EP0983091A1 (fr) * 1997-01-17 2000-03-08 Eli Lilly And Company Composition a base de proteines de l'obesite
WO2000021574A2 (fr) 1998-10-14 2000-04-20 Amgen Inc. Double polyethyleneglycolation de proteines dirigee sur site pour augmenter la bioactivite et la biocompatibilite
WO2000040615A2 (fr) * 1999-01-07 2000-07-13 Lexigen Pharmaceuticals, Corp. EXPRESSION ET EXPORTATION DE PROTEINES ANTI-OBESITE SOUS FORME DE PROTEINES HYBRIDE Fc
US6429290B1 (en) 1994-08-17 2002-08-06 The Rockefeller University OB polypeptides, modified forms and derivatives
US6541604B1 (en) 1996-01-08 2003-04-01 Genentech, Inc. Leptin receptor having a WSX motif
US6602694B1 (en) 1998-07-14 2003-08-05 Amylin Pharmaceuticals, Inc Uncoupling protein 4 (UCP-4)
US6620413B1 (en) 1995-12-27 2003-09-16 Genentech, Inc. OB protein-polymer chimeras
US6936439B2 (en) 1995-11-22 2005-08-30 Amgen Inc. OB fusion protein compositions and methods
US7074397B1 (en) 1996-01-08 2006-07-11 Genentech, Inc. Method for enhancing proliferation or differentiation of a cell using ob protein
WO2006096816A2 (fr) * 2005-03-09 2006-09-14 The University Of Pittsburgh Of The Commonwealth System Polypeptides derives de la leptine humaine et leurs utilisations
US7183254B2 (en) 2001-10-22 2007-02-27 Amgen, Inc. Use of leptin for treating human lipoatrophy and method of determining predisposition to said treatment
US7208577B2 (en) 1995-11-22 2007-04-24 Amgen, Inc. Methods of increasing lean tissue mass using OB protein compositions
US7524937B2 (en) 1996-01-08 2009-04-28 Genentech, Inc. WSX receptor agonist antibodies
US8076288B2 (en) 2004-02-11 2011-12-13 Amylin Pharmaceuticals, Inc. Hybrid polypeptides having glucose lowering activity
US8394765B2 (en) 2004-11-01 2013-03-12 Amylin Pharmaceuticals Llc Methods of treating obesity with two different anti-obesity agents
EP2286837A3 (fr) * 2004-11-01 2013-09-04 Amylin Pharmaceuticals, LLC Traitement de l'obésité et de maladies liés à l'obésité
US8926973B2 (en) 2001-03-30 2015-01-06 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
CN110128511A (zh) * 2019-05-31 2019-08-16 海南大学 一种用于减肥的重组蛋白、疫苗及其构建方法
US11535659B2 (en) 2010-09-28 2022-12-27 Amryt Pharmaceuticals Inc. Engineered polypeptides having enhanced duration of action

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US6001968A (en) * 1994-08-17 1999-12-14 The Rockefeller University OB polypeptides, modified forms and compositions
US7544492B1 (en) 1994-08-17 2009-06-09 The Rockefeller University OB polypeptides, modified forms and derivatives
US6429290B1 (en) 1994-08-17 2002-08-06 The Rockefeller University OB polypeptides, modified forms and derivatives
US7521258B2 (en) 1994-08-17 2009-04-21 The Rockefeller University Methods of detecting, measuring, and evaluating modulators of body weight in biological samples, and diagnostic, monitoring, and therapeutic uses thereof
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
US6025325A (en) * 1995-05-05 2000-02-15 Hoffman-La Roche Inc. Pegylated obese (ob) protein compositions
US8080254B2 (en) 1995-11-22 2011-12-20 Amgen, Inc. OB fusion protein compositions and methods
US7208577B2 (en) 1995-11-22 2007-04-24 Amgen, Inc. Methods of increasing lean tissue mass using OB protein compositions
US7112659B2 (en) 1995-11-22 2006-09-26 Amgen, Inc. OB fusion protein compositions and methods
US7718400B2 (en) 1995-11-22 2010-05-18 Amylin Pharmaceuticals, Inc. Methods of increasing lean tissue mass using OB protein compositions
US6936439B2 (en) 1995-11-22 2005-08-30 Amgen Inc. OB fusion protein compositions and methods
US6620413B1 (en) 1995-12-27 2003-09-16 Genentech, Inc. OB protein-polymer chimeras
US6541604B1 (en) 1996-01-08 2003-04-01 Genentech, Inc. Leptin receptor having a WSX motif
US7524937B2 (en) 1996-01-08 2009-04-28 Genentech, Inc. WSX receptor agonist antibodies
US7074397B1 (en) 1996-01-08 2006-07-11 Genentech, Inc. Method for enhancing proliferation or differentiation of a cell using ob protein
WO1997046585A2 (fr) * 1996-06-06 1997-12-11 Smithkline Beecham P.L.C. Fragments de leptine (proteine ob)
WO1997046585A3 (fr) * 1996-06-06 1998-04-23 Smithkline Beecham Plc Fragments de leptine (proteine ob)
WO1998028414A1 (fr) * 1996-12-20 1998-07-02 Eli Lilly And Company Proteines anti-obesite
WO1998028335A1 (fr) * 1996-12-20 1998-07-02 Eli Lilly And Company Proteines anti-obesite
EP0971742A4 (fr) * 1997-01-17 2002-04-10 Lilly Co Eli Formulations renfermant une proteine de l'obesite
EP0971742A1 (fr) * 1997-01-17 2000-01-19 Eli Lilly And Company Formulations renfermant une proteine de l'obesite
EP0983091A1 (fr) * 1997-01-17 2000-03-08 Eli Lilly And Company Composition a base de proteines de l'obesite
EP0983091A4 (fr) * 1997-01-17 2002-04-10 Lilly Co Eli Composition a base de proteines de l'obesite
WO1998041222A1 (fr) * 1997-03-20 1998-09-24 Eli Lilly And Company Formulations de proteine de l'obesite
US6602694B1 (en) 1998-07-14 2003-08-05 Amylin Pharmaceuticals, Inc Uncoupling protein 4 (UCP-4)
WO2000021574A2 (fr) 1998-10-14 2000-04-20 Amgen Inc. Double polyethyleneglycolation de proteines dirigee sur site pour augmenter la bioactivite et la biocompatibilite
US6420339B1 (en) 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
WO2000040615A2 (fr) * 1999-01-07 2000-07-13 Lexigen Pharmaceuticals, Corp. EXPRESSION ET EXPORTATION DE PROTEINES ANTI-OBESITE SOUS FORME DE PROTEINES HYBRIDE Fc
WO2000040615A3 (fr) * 1999-01-07 2000-11-23 Lexigen Pharm Corp EXPRESSION ET EXPORTATION DE PROTEINES ANTI-OBESITE SOUS FORME DE PROTEINES HYBRIDE Fc
US8926973B2 (en) 2001-03-30 2015-01-06 Merck Patent Gmbh Reducing the immunogenicity of fusion proteins
US8318666B2 (en) 2001-10-22 2012-11-27 Amgen, Inc. Use of leptin to treat metabolic abnormalities associated with lipoatrophy
US7183254B2 (en) 2001-10-22 2007-02-27 Amgen, Inc. Use of leptin for treating human lipoatrophy and method of determining predisposition to said treatment
EP2219031A1 (fr) 2001-10-22 2010-08-18 Amgen, Inc. Utilisation de Leptine pour traiter la Lipoatrophine humaine et procédé pour déterminer une prédisposition à ce traitement
US9453063B2 (en) 2004-02-11 2016-09-27 Amylin Pharmaceuticals, Llc. Hybrid polypeptides with selectable properties
US8697647B2 (en) 2004-02-11 2014-04-15 Odile Esther Levy Hybrid polypeptides with selectable properties
US8076288B2 (en) 2004-02-11 2011-12-13 Amylin Pharmaceuticals, Inc. Hybrid polypeptides having glucose lowering activity
EP2286837A3 (fr) * 2004-11-01 2013-09-04 Amylin Pharmaceuticals, LLC Traitement de l'obésité et de maladies liés à l'obésité
EP1814590B2 (fr) † 2004-11-01 2013-12-11 Amylin Pharmaceuticals, Inc. Traitement contre l'obesite ainsi que les maladies associees a l'obesite
US8394765B2 (en) 2004-11-01 2013-03-12 Amylin Pharmaceuticals Llc Methods of treating obesity with two different anti-obesity agents
EP2286840A3 (fr) * 2004-11-01 2013-09-04 Amylin Pharmaceuticals, LLC Traitement de l'obésité et de maladies liés à l'obésité
EP2286838A3 (fr) * 2004-11-01 2013-09-04 Amylin Pharmaceuticals, LLC Traitement de l'obésité et de maladies liés à l'obésité
EP2286839A3 (fr) * 2004-11-01 2013-09-04 Amylin Pharmaceuticals, LLC Traitement de l'obésité et de maladies liés à l'obésité
EP2127676A3 (fr) * 2004-11-01 2013-09-04 Amylin Pharmaceuticals, LLC Traitement de l'obésité et les maladies et troubles liés à l'obésité
JP2008535797A (ja) * 2005-03-09 2008-09-04 ザ ユニバーシティ オブ ピッツバーグ オブ ザ コモンウェルス システム ヒトレプチン由来のポリペプチドとその使用
WO2006096816A2 (fr) * 2005-03-09 2006-09-14 The University Of Pittsburgh Of The Commonwealth System Polypeptides derives de la leptine humaine et leurs utilisations
US7629315B2 (en) 2005-03-09 2009-12-08 University Of Pittsburgh Of The Commonwealth System Of Higher Education Compositions for blocking the inhibitory effect of human CRP on human leptin
WO2006096816A3 (fr) * 2005-03-09 2007-05-24 Univ Pittsburgh Polypeptides derives de la leptine humaine et leurs utilisations
US11535659B2 (en) 2010-09-28 2022-12-27 Amryt Pharmaceuticals Inc. Engineered polypeptides having enhanced duration of action
CN110128511A (zh) * 2019-05-31 2019-08-16 海南大学 一种用于减肥的重组蛋白、疫苗及其构建方法
CN110128511B (zh) * 2019-05-31 2022-10-25 海南大学 一种用于减肥的重组蛋白、疫苗及其构建方法

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