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WO1997016550A1 - Fragments polypeptides derives du produit genique obese - Google Patents

Fragments polypeptides derives du produit genique obese Download PDF

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Publication number
WO1997016550A1
WO1997016550A1 PCT/US1996/017365 US9617365W WO9716550A1 WO 1997016550 A1 WO1997016550 A1 WO 1997016550A1 US 9617365 W US9617365 W US 9617365W WO 9716550 A1 WO9716550 A1 WO 9716550A1
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WIPO (PCT)
Prior art keywords
seq
obf
polypeptide
sequence
diabetes
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PCT/US1996/017365
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English (en)
Inventor
Claudio Mapelli
Chester A. Meyers
Stanley R. Krystek, Jr.
Jiri Novotny
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Bristol-Myers Squibb Company
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Publication of WO1997016550A1 publication Critical patent/WO1997016550A1/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 concerns polypeptide fragments derived from the obese (ob) gene product and uses thereof.
  • the mouse and human ob gene were recently identified by positional cloning and sequencing (Friedman et al. Nature 372, 425-432 (1994)).
  • ob encodes a 4.5 kb adipose tissue messenger RNA containing a predicted 167-amino acid open reading frame (ORF).
  • ORF predicted amino acid sequence is 84% identical between human and mouse and has features of a secreted protein.
  • the extensive homology deduced for the ob gene product among vertebrates suggests that its function is highly conserved.
  • the present invention is directed to polypeptide fragments of the obese gene product which have anti-obesity and/or anti-diabetic activity. These polypeptides will be referred to herein as the active obese gene product fragments or "OBF" which will be further defined hereinafter.
  • OBF active obese gene product fragments
  • the invention also includes therapeutic methods using OBF. Accordingly, in a first embodiment, the invention is directed to purified preparations of OBF.
  • a preferred murine OBF has the sequence of SEQ.ID.NO.:1 as follows:
  • a preferred human OBF has the sequence of SEQ.ID.NO.:2 as follows: Leu-Glu-Asn-Leu-Arg-Asp-Leu-Leu-His-Leu-Leu-Ala-Phe-Ser-Lys-Ser-Cys-Ser-Leu- Pro-Gln-Thr-Ser-Gly-Leu-Gln-Lys-Pro-Glu-Ser-Leu-Asp-Gly-Val-Leu-Glu-Ala-Ser- Leu-Tyr-Ser-Thr-Glu-Val-Val-Ala-Leu-Ser-Arg-Leu-Gln-Gly-Ser-Leu-Gln-Asp-lle-Leu- Gln-Gln-Leu-Asp-Val-Ser-Pro-Glu-Cys, S,S-crosslinked A preferred human OBF has the sequence of SEQ.ID.NO.:2 as follows: Leu-Glu-
  • Another preferred murine OBF has the sequence of SEQ.ID.NO.:3 as follows: Ser-Lys-Ser-Cys-Ser-Leu-Pro-Gln-Thr-Ser-Gly-Leu-Gln-Lys-Pro-Glu-Ser-Leu-Asp- Gly-Val-Leu-Glu-Ala-Ser-Leu-Tyr-Ser-Thr-Glu-Val-Val-Ala-Leu-Ser-Arg-Leu-Gln- Gly-Ser-Leu-Gln-Asp-lle-Leu-Gln-Gln-Leu-Asp-Val-Ser-Pro-Glu-Cys, S,S-crosslinked
  • Another preferred human OBF has the sequence of SEQ.ID.NO.:4 as follows: Ser-Lys-Ser-Cys-His-Leu-Pro-Trp-Ala-Ser-Gly-Leu-Glu-Thr-Leu-Asp-Ser-Leu-Gly- Gly-Val-Leu-Glu-Ala-Ser-Gly-Tyr-Ser-Thr-Glu-Val-Val-Ala-Leu-Ser-Arg-Leu-Gln- Gly-Ser-Leu-Gln-Asp-Met-Leu-Trp-Gln-Leu-Asp-Leu-Ser-Pro-Gly-Cys, S,S- crosslinked
  • the invention is directed to a method for treating obesity in a patient , wherein the obesity is related to a defect in regulation by the obese protein or can otherwise be overcome by elevating obese protein levels, the method comprising administering an effective amount of OBF to the patient.
  • the invention is directed to a method for treating diabetes in a patient in need of treatment, where the diabetes is obese protein - associated, the method comprising administering an effective amount of OBF to the patient.
  • the invention is directed to a method for diagnosing obese protein-associated diabetes or obesity.
  • the invention is directed to an isolated nucleic acid sequence encoding OBF.
  • the invention is directed to a vector containing nucleic acid encoding OBF.
  • the invention is directed to a host cell transformed with the vector of the invention.
  • the invention is directed to a method for producing OBF from the recombinant host.
  • Figure 1 Three dimensional representation of preferred OBF having 67 amino acids and three alpha helices. It has been discovered that fragments of the ob protein retaining biological activity and accessible by chemical synthesis overcome some of the drawbacks of the full length protein. Specifically, it has been found by secondary structure analysis and molecular modeling that the C-terminal peptide fragments containing the only two cysteine residues present in the mature ob protein, which are preferably disulfide- linked (or functionally equivalent residues that are capable of forming cross-linkages), retains the conformation and overall folding needed for biological activity.
  • therapeutic methods means the anti-obesity and/or the anti-diabetic methods of the invention
  • OFB means the optionally mutated or modified biologically active C-terminal fragments of the ob gene product having less than sixty-eight amino acids and containing two residues at positions 117 and 167 that are capable of forming cross ⁇ linkages , preferably cysteine residues
  • obese protein-associated means that the obesity and or diabetes is partially or wholly due to mutations of the ob gene or any alteration or defect in the ob gene product or metabolic pathway(s) involving the ob gene product resulting in increased risk or clinical manifestation of obesity and/or diabetes
  • biologicalcally active means having serum glucose lowering activity useful for treating type II diabetes; or serum triglyceride lowering activity, or decreased body weight gain, or decreased body weight, or reduction in food consumption or appetite useful for treating obesity.
  • OBF preferably has 67 amino acids and most preferably has the amino acid sequence identified as SEQ. ID. NO:1 or SEQ.ID.NO.:2; however, other biologically active C-terminal fragments of the ob gene product having less than 68 amino acids are also specifically contemplated to be within the scope of the present invention.
  • the amino acid numbering scheme used herein is based on the intact ob protein and is the same as described by Friedman and coworkers in Nature 372, 425-432 (1994).
  • Such fragments of the invention which include mutants or modified fragments that retain biological activity, contain amino acid sequences with the following typical features: (1) residues in positions 117 and 167 with side-chains capable of forming cross-linkages such as disulfide, lactam, lactone, dicarba-cystine and the like; (2) an amphipathic alpha-helix spanning about 15-25 C-terminal residues; (3) a preferred but optional amphipathic alpha-helix N-terminal to the Cys 117 or equivalent residue, spanning about 15-25 residues; (4) a loop supported by at least one helical scaffold and at least one covalent cross-linkage.
  • OBF containing one additional cross-linkage between the C-terminal alpha-helix and either the loop or the N-terminal alpha-helix, if present are specifically contemplated to be within the scope of the present invention.
  • the present invention specifically contemplates polypeptides that are at least 30% homologous to the polypeptides of SEQ.ID.NO.:1 , SEQ.ID.N0.2, SEQ.ID.NO.:3, or SEQ.ID.NO. :4, preferably at least 50% homologous, more preferably at least 80% homologous, and most preferably at least 90% homologous.
  • Residues 117 and 167 are preferably cross-linked via covalent bonds such as disulfide, lactam, lactone, dicarba-cystine and the like.
  • the OBF preferably has cysteine residues at positions 117 and 167 which are preferably cross-linked via disulfide bonds.
  • the N- terminus of such fragments is typically blocked with an acetyl or similar N-blocking group.
  • OBF can be prepared by chemical synthesis using the following exemplary process:
  • NMP N- methylpyrrolidinone
  • DMF dimethylformamide
  • a coupling reagent such as 2-(1 H-benzotriazol-1 -yl)-1 , 1 ,3,3- tetramethyluronium hexafluorophate (HBTU) and 1-hydroxybenzotriazole (HOBt) in NMP and diisopropylethylamine (DIEA) at about 20 to 30 °C in a molar ratio of about 1: 5 compound l:ll to form a resin-coupled dipeptide (III)
  • a coupling reagent such as 2-(1 H-benzotriazol-1 -yl)-1 , 1 ,3,3- tetramethyluronium hexafluorophate (HBTU) and 1-hydroxybenzotriazole (HOBt) in NMP and diisopropylethylamine (DIEA) at about 20 to 30 °C in a molar ratio of about 1: 5 compound l:ll to form a resin-coupled dipeptide (III)
  • P 1 is an amino-protecting group such as flourenylmethoxycarbonyl (Fmoc) and P 2 is a para-alkoxybenzyl ester linkage.
  • the reaction may be effected with an automated peptide synthesizer (e.g., Applied Biosystems 431A or 433A.).
  • the foregoing procedure for peptide III is repeated with other nitrogen-protected and side-chain protected amino acids until all desired residues, e.g.,of SEQ. ID. NO. 1 or SEQ. ID. NO. 2, have been coupled to the resin.
  • Sidechain-protecting groups may be used in this process for sidechains having reactive functionalities, such as hydroxyl, carboxyl, amino, mercapto, guanidino, imidazolyl, indolyl and the like.
  • reactive functionalities such as hydroxyl, carboxyl, amino, mercapto, guanidino, imidazolyl, indolyl and the like.
  • the particular protecting groups used for any amino acid residues depend upon the sidechains to be protected and are generally in the art.
  • Exemplary sidechain protecting groups are t-butyl, benzyl, benzoyl, acetyl, halocarbobenzoxy, and the like for hydroxyl; cyclohexyl, benzyl, methyl, ethyl, f-butyl and the like for carboxyl; benzyl, 4-methylbenzyl, 4-methoxybenzyl, acetyl, acetamidomethyl, triphenylmethyl (trityl), and the like for mercapto; i- butyloxycarbonyl (Boc), carbobenzoxy (Cbz), halocarbobenzoxy, 9- flourenylmethoxycarbonyl (Fmoc), phthaloyl (Pht), p-toluenesulfonyl (Tos), trifluoroacetyl, 2-(trimethylsiiyl)ethoxycarbonyl (Teoc), and the like for amino; 2,4
  • Side chain protecting groups may be removed, if desired, by treatment with one or more deprotecting agents in an inert solvent or solvent mixture (e.g., NMP or DMF).
  • Suitable deprotecting agents are generally known in the art. Exemplary deprotecting agents are thiophenol, mercaptoethanol and the like for removing 2,4- dinitrophenyl; trifluoroacetic acid (TFA) and the like for butoxycarbonyl; hydrofluoric acid (HF), trifluoromethanesulfonic acid (TFMSA) and the like for several different protecting groups.
  • TFA trifluoroacetic acid
  • HF hydrofluoric acid
  • TFMSA trifluoromethanesulfonic acid
  • the fully formed polypeptide may be cleaved from the resin by methods generally known in the art.
  • the resin-bound polypeptide may be treated with an acid, such as TFA, HF, TFMSA, and the like.
  • Cross-linkage of the two cysteine residues via disulfide bond may be effected by methods generally known in the art.
  • the deprotected peptide may be stirred overnight in 0.1 M ammonium bicarbonate (pH 7.8) or in dilute ammonium hydroxide (pH 8.0-8.5) in the presence or not of 10% dimethylsulfoxide.
  • the crude peptide may be dissolved in a solution of water/acetonitrileTFA (60:40:0.1, v:v:v) and loaded onto a C18 reverse phase column.
  • a gradient of acetonitrile in water, both buffered with 0.1% TFA, can then be used to elute the desired OBF.
  • OBF also can be prepared by use of genetic engineering techinques.
  • the process for genetically engineering the OBF coding sequence, for expression under a desired promoter, is facilitated through the cloning of genetic sequences which are capable of encoding OBF.
  • These cloning technologies can utilize techniques known in the art for construction of a DNA sequence encoding OBF, such as polymerase chain reaction technologies utilizing a OBF sequence to isolate the ob gene de novo , or polynucleotide synthesis methods to construct the nucleotide sequence using chemical methods. Expression of the cloned OBF DNA provides OBF.
  • genetic sequences is intended to refer to a nucleic acid molecule (preferably DNA). Genetic sequences that are capable of being operably linked to DNA encoding OBF, so as to provide for its expression and maintenance in a host cell, are obtained from a variety of sources, including commercial sources, genomic DNA, cDNA, synthetic DNA, and combinations thereof. Since the genetic code is universal, it is to be expected that any DNA encoding the OBF amino acid sequence of the invention will be useful to express OBF in any host, including prokaryotic (bacterial) and eukaryotic (plants, mammals (especially human), insects, yeast, and especially cultured cell populations).
  • prokaryotic bacterial
  • eukaryotic plants, mammals (especially human), insects, yeast, and especially cultured cell populations.
  • mice DNA sequence encoding OBF is SEQ.ID.NO.:5 as follows: CCTGGAGAATCTCCGAGACCTCCTCCATCTGCTGGCCTTCTCCAAGA GCTGCTCCGCCTCAGACCAGTGGCCTGCAGAAGCCAGAGAGCCTGGA TGGCGTCCTGGAAGCCTCACTCTACTCCACAGAGGTGGTGGCTTTGA GCAGGCTGCAGGGCTCTCTGCAGGACATTCTTCAACAGTTGGATGTT AGCCCTGAATG
  • mice DNA sequence encoding OBF is SEQ.ID.NO.:6 as follows: CTCCAAGAGCTGCTCCGCCTCAGACCAGTGGCCTGCAGAAGCCAGAG AGCCTGGATGGCGTCCTGGAAGCCTCACTCTACTCCACAGAGGTGGT GGCTTTGAGCAGGCTGCAGGGCTCTCTCTGCAGGACATTCTTCAACAGT TGGATGTTAGCCCTGAATG
  • An example of human DNA sequence encoding OBF is SEQ.ID.N0.7 as follows: CTGGAGAACCTCCGGGATCTTCTTCACGTGCTGGCCTTCT CTAAGAGCTGCCACTTGCCCTGGGCCAGTGGCCTGGAGACCTTGGAC AGCCTGGGGGGTGTCCTGGAAGCTTCAGGCTACTCCACAGAGGTGGT GGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAGC TGGACCTCAGCCCTGGGTGC TGGACCTCAGCCCTGGGTGC TGGACCTCAGCCCTGGGTGC TGGACCTCAGCCCTGGGT
  • SEQ.ID.NO.:8 Another example of human DNA sequence encoding OBF is SEQ.ID.NO.:8 as follows: TCTAAGAGCTGCCACTTGCCCTGGGCCAGTGGCCTGGAGACCTTGGA CAGCCTGGGGGGTGTCCTGGAAGCTTCAGGCTACTCCACAGAGGTGG TGGCCCTGAGCAGGCTGCAGGGGTCTCTGCAGGACATGCTGTGGCAG CTGGACCTCAGCCCTGGGTGC
  • DNA sequences which encode the same amino acid sequences of SEQ.ID.NOS.: 1 ,2,3, and 4 may be used for the production of the OBF of the present invention.
  • allelic variations of these DNA and amino acid sequences naturally exist, of may be intentionally introduced using methods known in the art. These variations may be demonstrated by one or more amino acid acid differences in overall sequence, or by deletions, substitutions, insertions, inversions or additions of one or more amino acids in said sequence.
  • Such amino acid substitutions may be made, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphiphathic nature of the residues involved.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and agrginine; amino acids with uncharged polar head groups or nonpolar head groups having similar hydrophilicity values include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine.
  • Other contemplated variations include salts and esters of the aforementioned polypeptides, as well as precursors of the aformentioned polypeptides, for example, precursors having N-terminal substitutions such as methionine, N-formylmethionine and leader sequences. All such variations are included within the scope of the pressent invention.
  • the present invention include DNA sequences that code for biologically active polypeptides of less than 68 amino acids and that are capable of hybridizing under stringent conditions to a sequence complementary to SEQ.ID.NOS.: 5, 6, 7, or 8.
  • Stringent hybridization conditions select for DNA sequences of greater than 30% homology, preferably greater than 85% or, more preferably, greater than 90% homology. Screening DNA under stringent conditions may be carried out according to the method described in Nature. 313: 402- 404 (1985).
  • the DNA sequences capable of hybridizing under stringent conditions with the DNA disclosed in the present application may be, for example, allelic variants of the disclosed DNA sequences, may be naturally present in the human or mouse but related to the disclosed DNA sequences, or may be derived from other animal sources.
  • the expression of the encoded OBF can occur through the transient (nonstable) expression of the introduced sequence.
  • the coding sequence is introduced on a DNA molecule, such as a closed circular or linear molecular that is capable of autonomous replication. If integration into the host chromosome is desired, it is preferable to use a linear molecule. If stable maintenance of OBF DNA is desired on an extrachromosomal element, then it is preferable to use a circular plasmid form, with appropriate plasmid element for autonomous replication in the desired host.
  • the desired gene construct providing DNA coding for OBF, and the necessary regulatory elements operably linked thereto, can be introduced into desired host cells by transformation, transfection, or any method capable of providing the construct to the cell.
  • a marker gene for the detection of a host cell that has accepted the OBF DNA can be on the same vector as the OBF DNA or on a separate construct for co-transformation with the OBF coding sequence construct into the host cell.
  • the nature of the vector will depend on the host organism. Suitable selection markers will depend upon the host cell.
  • the marker can provide biocide resistance, e.g, resistance to antibiotics, or heavy metals, such as copper, or the like.
  • Factors of importance in selecting a particular plasmid or viral vector include: the ease with which recipient cells that contain the vector can be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells or different species.
  • Oligonucleotide probes specific for the OBF sequence can be used to identify clones to OBF and can be designated de novo from the knowledge of the amino acid sequence of the protein as provided herein in SEQ.ID.NO.:1 or from the knowledge of the nucleic acid sequence of the DNA encoding such protein or of a related protein.
  • antibodies can be raised against OBF and used to identify the presence of unique protein determinants in transformants that express the desired cloned polypeptide.
  • a nucleic acid molecule, such as DNA is said to be "capable of expressing"
  • nucleic acid if that nucleic acid contains expression control sequences which contain transeriptional regulatory information and such sequences are "operably linked" to the OBF nucleotide sequence which encode the OBF polypeptide.
  • An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (or sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence.
  • the two DNA sequences are a coding sequence and a promoter region sequence linked to the 5' end of the coding sequence, they are operably linked if induction of promoter function results in the transcription of mRNA encoding the desired protein and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the expression regulatory sequences to direct the expression of the protein, antisense RNA, or (3) interfere with the ability of the DNA template to be transcribed.
  • a promoter region is operably linked to a DNA sequence if the promoter is capable of effecting transcription of that DNA sequence.
  • the precise nature of the regulatory regions needed for gene expression can vary between species or cell types, but includes, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively, such as the TATA box, capping sequence, CAAT sequence, and the like, with those elements necessary for the promoter sequence being provided by the promoters of the invention.
  • transeriptional control sequences can also include enhancer sequences or upstream activator sequences, as desired.
  • the vectors of the invention can further comprise other preferably linked regulatory elements such as DNA elements which confer antibiotic resistance, or origins of replication for maintenance of the vector in one or more host cells.
  • Expression of a protein in eukaryotic hosts such as a human cell requires the use of regulatory regions functional in such hosts.
  • a wide variety of transeriptional and translational regulatory sequences can be employed, depending upon the nature of the host.
  • these regulatory signals are associated in their native state with a particular gene which is capable of a high level of expression in the specific host cell, such as specific human tissue type.
  • control regions may or may not provide an initiator methionine (AUG) codon, depending on whether the cloned sequence contains such a methionine.
  • AUG initiator methionine
  • Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell.
  • the non-transcribed and/or non-translated regions 3' to the sequence coding for ob protein can be obtained by the above-described cloning methods.
  • the 3'-non-transcribed region of the native human obgene can be retained for its transeriptional termination regulatory sequence elements, or for those elements which direct polyadenylation in eukaryotic cells. Where the native expression control sequences signals do not function satisfactorily in a host cell, sequences functional in the host cell can be substituted.
  • a fusion production that contains a partial coding sequence (usually at the amino terminal end) of a first protein or small peptide and a second coding sequence (partial or complete) of OBF at the carboxyl end.
  • the coding sequence of the first protein can, for example, function as a signal sequence for secretion of OBF from the host cell.
  • Such first protein can also provide for tissue targeting or localization of OBF if it is to be made in one cell type in a multicellular organism and delivered to another cell type in the same organism.
  • Such fusion protein sequences can be designed with or without specific protease sites such that a desired peptide sequence is amenable to subsequent removal.
  • the expressed OBF can be isolated and purified from the medium of the host in accordance with conventional conditions, such as extraction, precipitation, ion exchange chromatography, size exclusion chromatography, affinity chromatography, high performance liquid chromatography on reverse phase, electrophoresis, or the like.
  • affinity purification with anti-OBF antibody can be used.
  • a polypeptide having the amino acid sequence of, for example,SEQ.ID.NO.:1 or SEQ.ID.NO.:2 can be made, or a shortened peptide of either of these sequences can be made, and used to raise antibodies using methods well known in the art. These antibodies can be used to affinity purify or quantitate OBF from any desired source.
  • the host cells can be collected by centrifugation, or with suitable buffers, lysed, and the protein isolated by the column chromatography, for example, on DEAE- cellulose, phosphocellulose, polyribocytidylc acid-agarose, hydroxyapatite or by electrophoresis or immunoprecipitation.
  • Mouse ob coding sequence can be amplified using the polymerase chain reaction (PCR) from mouse adipose tissue cDNA using synthetic oiigonucleotides corresponding to the 5' and 3' ends of the 501 nucleotide coding sequence (Friedman et al. Nature 372, 425-432 (1994)), and cloned into the £.
  • PCR polymerase chain reaction
  • coli expression vector pGEX- 2T (Pharmacia) to produce a C-terminal glutathione-S-transferase fusion protein coding sequence.
  • Further constructs could be made that would allow heterologous ob protein expression in baculovirus-infected insect cells, COS cells, 293 cells, and Saccaromyces cerevisia by cloning the above ob coding sequence into the vectors pVL1393, pCDNA1/amp, pCEP4 ⁇ , and a suitable Saccaromyces cerevisia vector, respectively. It is understood that although the following discussion is specifically directed to human patients, the teachings are also applicable to any animal which would benefit from pracitce of the methods of the invention.
  • OBF preferably of the formula SEQ. ID. NOS.: 1, 2, 3, or 4
  • SEQ. ID. NOS.: 1, 2, 3, or 4 may be used to reduce serum glucose and/or triglyceride levels in obese patients and type II diabetics.
  • Plasma glucose and triglyceride levels are significantly decreased in oblob mice, following infusion with OBF, relative to infusion with a control peptide.
  • the anti-diabetic effect is independent of an effect on food consumption.
  • the form of administration of OBF can be any form known in the pharmaceutical art.
  • the amount of OBF to be administered would depend in part on the age, weight, and general condition of the patient. Typically, a patient would be closely monitored by a physician who would determine if the dosage amount or regimen of OBF being administered was effective and well tolerated.
  • OBF would be administered either alone or admixed with a pharmaceutically acceptable carrier. Administration can be parenteral or enteral depending upon the dosage form and the needs of the patient.
  • the effective amount of OBF in either the anti-obesity or anti-diabetic methods of the invention is typically about 0.001 to about 1000 milligrams (mgs) per kilogram (kg) of body weight (wt) per day, preferably about 0.05 to about 50 mgs per kg of body weight per day. It is another advantage of the present invention that activity-retaining radioisotopes of OBF are easier to prepare than those of corresponding full length protein.
  • the OBF polypeptide defined by SEQ. ID. NOS.:1 , 2, 3, or 4 may be labeled with a radioisotope including radioactive iodo ( 125 l) or hydrogen ( 3 H) atoms.
  • labeling of the OBF polypeptide defined by SEQ. ID. NOS.:1, 2, 3, or 4 with radioactive iodo ( 125 l) atoms may be achieved by reaction with prelabeled Bolton-Hunter reagent.
  • a radiolabelled OBF e.g., the polypeptide defined by SEQ. ID. NOS.: 1, 2, 3, or 4
  • the present invention is also directed to a method of diagnosing obesity or diabetes or a predisposition to develop obesity or diabetes in an individual, said method comprising
  • step (d) comparing the first set of results from step (c) with with a second set of results obtained using similar monitoring in an individual not having OBF-associated obesity or diabetes;
  • the labelling can be any type of labelling known in the art such as radiolabelling, fluorescent labelling, biotinylation, and the like.
  • OBF of the present invention may also be used to prepare in a known manner polyclonal or monoclonal antibodies capable of binding the OBF. These antibodies may in turn be used for the detection of the ob protein in a sample, for example, a plasma sample or cell sample, using immunoassay techniques, for example, radioimmunoassay or enzyme immunoassay. The antibodies may also be used in affinity chromatography for purifying OBF or ob protein and isolating it from various sources.
  • the present invention is also directed to a method of diagnosing obesity or diabetes or a predisposition to develop obesity or diabetes in an individual, said method comprising (a) raising antibodies to OBF in a suitable mammal such as mouse, rabbit, horse.etc,
  • SEQ. ID. NO.-.1 N-Acetyl-L-leucyl-L- ⁇ -glutamyl-L-asparaginyl-L-leucyl-L-arginyl-L- ⁇ -aspartyl-L- leucyl-L-leucyl-L-histidyl-L-leucyl-L-leucyl-L-alanyl-L-phenylalanyl-L-seryl-L-lysyl-L- seryl-L-cysteinyl-L-seryl-L-leucyl-L-prolyl-L-glutaminyl-L-threonyl-L-serylglycyl-L- leucyl-L-glutaminyl-L-lysyl-L-prolyl-L- ⁇ -glutamyl-L-seryl-L-leucyl-L- ⁇ -a
  • a protected peptide ("intermediate A") of SEQ.ID.NO.:1 was assembled stepwise by the solid phase method on an Applied Biosystems Model 431 A Peptide Synthesizer using the Fmoc/HBTU/HOBt program supplied with the instrument.
  • the starting Fmoc-Cys(Trt)-HMP resin polystyrene, 1% divinylbenzene copolymer
  • Amino acids were coupled as their N ⁇ -Fmoc derivatives.
  • the side chains of Ser, Thr and Tyr were protected with the t-butyl group; those of Cys, Asn, His and Gin with the trityl group, those of Asp and Glu with the t-butyl ester group, that of Arg with the 2,2,5,7,8- pentamethylchroman-6-sulfonyl (PMC) group and that of Lys with the N ⁇ -t- butyloxycarbonyl (t-Boc) group. Fmoc groups were removed at each cycle by a 6 min treatment with 20% piperidine in NMP.
  • Each amino acid derivative was double coupled (25 min, room temperature), in 5-fold molar excess, using one equivalent of 2- (1H-benzotriazol-1-yl)-1 ,1,3,3-tetramethyluronium hexafluorophate (HBTU) and 1- hydroxybenzotriazole (HOBt) in NMP and DIEA.
  • HBTU 2- (1H-benzotriazol-1-yl)-1 ,1,3,3-tetramethyluronium hexafluorophate
  • HOBt 1- hydroxybenzotriazole
  • Example 1 As a white, fluffy solid.
  • a protected peptide ("intermediate B") of SEQ.ID.NO.:3 was assembled stepwise by the solid phase method on an Applied Biosystems Model 431 A Peptide Synthesizer using the Fmoc/HBTU/HOBt program supplied with the instrument.
  • the starting Fmoc-Cys(Trt)-HMP resin polystyrene, 1% divinylbenzene copolymer
  • Amino acids were coupled as their N ⁇ -Fmoc derivatives.
  • the side chains of Ser, Thr and Tyr were protected with the t-butyl group; those of Cys and Gin with the trityl group, those of Asp and Glu with the t-butyl ester group, that of Arg with the 2,2,5,7,8- pentamethylchroman-6-sulfonyl (PMC) group and that of Lys with the N ⁇ -t- butyloxycarbonyl (t-Boc) group.
  • Fmoc groups were removed at each cycle by a 6 min treatment with 20% piperidine in NMP.
  • Each amino acid derivative was double coupled (25 min, room temperature), in 5-fold molar excess, using one equivalent of 2- (1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophate (HBTU) and 1- hydroxybenzotriazole (HOBt) in NMP and DIEA.
  • HBTU 2- (1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophate
  • HOBt 1- hydroxybenzotriazole
  • Example 2 The fractions containing the major peak (215 nm) eluting between 27 and 30 min were pooled and lyophilized to yield 16 mg (20%) of Example 2 as a white, fluffy solid.
  • Analytical HPLC of the above-prepared OBF Vydac C18 (4.6 x 250 mm); 215 nm, 1.0 mlJmin: linear gradient from 35% to 45% B in A over 50 minutes; retention time 30.63 minutes. Purity: greater than 95% (Solvent A, 0.1% TFA in water; Solvent B, 0.1% TFA in acetonitrile). MS (electrospray): found molecular weight (derived) 5788.6.
  • the OBFs can be expressed in £ coli as maltose binding fusion proteins (MBP) using an expression vector from New England Biolabs (pMALTM-c, 1990).
  • MBP maltose binding fusion proteins
  • the OBFs are ampified by PCR and cloned as Asp718/Sal I fragments by replacing most of the multiple cloning site and the factor Xa site of this vector.
  • a two-step PCR protocol is used to simultaneously introduce a protease site (thrombin site) and a unique restriction enzyme site virtually anywhere within the OBFs.
  • thrombin site downstream of Asn 99 and simultaneously a BamHI restriction enzyme site (bold) are introduced, resulting in the following construction: 95 99 100
  • GAG AAT SEQ.ID.NO. :9
  • the DNA and protein sequences of the ob gene construct containing a thrombin site within the gene are given in the one-letter code.
  • the numbering refers to the amino acid numbering of the ob protein.
  • Bold is the recognition sequence for the BamHI restriction enzyme.
  • the DNA sequence in italic derives from the ob gene.
  • step 1a template full length ob gene primer MAspNN and Nov2rev
  • step 1b template full length ob gene primer Nov2for
  • step 2 templates products of PCR reactions 1a and 1b primers MAspNN and MSalCC
  • MAspNN 5 '- TCG GTA CCG GTG CCT ATC CAG AAA (SEQ.IN.NO. :11)
  • This system can be used for any modification within the gene by just changing the matching sequences in oiigonucleotides Nov2rev and Nov2for.
  • a second strategy to obtain the construct shown in figure 1 is as follows.
  • the ob gene is amplified as BamHI/Sal I fragment (using oiigonucleotides MBamNN 5'- GGATCC GTGCCTATCCAGAAA GTC (SEQ.ID.NO.:15) and MSalCC 5'-
  • mutagenesis vector system i.e. pALTER-1 from Promega, 1994.
  • the in vitro mutagenesis is performed as described by the manufacturer using the Nov2for oligonucleotide (see above).
  • the mutated ob gene is cloned into the pMALTM. c expression vector (New England Biolabs, 1990). This mutagenesis system also allows introduction of a thrombin site into the ob gene at any other location by changing the matching sequences in oligonucleotide Nov2for.
  • the cell pellet (from 1 to 1.5 L cell culture) is resuspended in 30 mL lysis buffer (10 mM MOPS/NaOH pH 6.0, 2 mM EDTA, 500 mM NaCl, protease inhibitors) in a homogenizer and lysed by french press (SLM Aminco 40k preparative cell, single run at 1200 PSI).
  • the lysis suspension is centrifuged (35 min, 35,000 rpm, Beckman 45TI rotor).
  • the fusion protein is in the supernatant and can be directly affinity purified on 800-21 amylose resin (Biolabs Inc.) following the manufacturer's instructions.
  • the protein is eluted in the lysis buffer containing 50 mM maltose.
  • the pH of the eluate is increased to pH 8-9 and bovine thrombin (Amour Pharmaceutical Compny) is added to 5 U/mg fusion protein.
  • the reaction is incubated for 30-40 min at RT and stopped with 1 mM benzamidine.
  • the OBFs are precipitated with 40% acetone, oxidized and further purified by reversed phase HPLC as described above.
  • mice Female obese (ob/ob) mice were obtained from Jackson Laboratory (Bar Harbor, ME) at 5-6 weeks of age. The animals were housed two per cage at 24°C with a 6 am to 6 pm light cycle. They were give ad lib access to water and powdered food (Purina 9F-5020) in food cups. Mice were acclimated for two weeks prior to implantation of the Alzet pumps.
  • Plasma chemistries were determined enzymatically using a Roche COBAS-MIRA S clinical analyzer. Plasma glucose and triglyceride levels were determined by an assay from Roche Diagnostics, Montclair, NJ; plasma non-esterified fatty acids (NEFA) levels were quantitated by an assay obtained from Wako Biochemicals, Wako, TX. Plasma insulin levels were determined by a radioimmunoassay (Incstar Corporation, Stillwater, MN), utilizing rat insulin as the standard. Plasma corticosterone levels were determined by a double antibody radioimmunoassay (ICN Biochemicals, Costa Mesa, CA) utilizing rat corticosterone as the standard.
  • BAT brown adipose tissue
  • Plasma TG (mg/dl) 101 ⁇ 4 81 ⁇ 6 * (-20%)
  • Plasma NEFA (mEq/L) 1.15 ⁇ 0.06 1.36 ⁇ 0.02" (+18%)
  • Plasma Insulin (ng/ml) 3.49 + 0.39 4.56 ⁇ 0.86

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  • Child & Adolescent Psychology (AREA)
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  • Obesity (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Nouveaux fragments C-terminaux du produit génique obèse. Ces fragments conservent des activités dirigées contre l'obésité et/ou le diabète. Lesdits fragments comprennent moins de 68 acides aminés, dont deux sont aptes à former des réticulations, de préférence des cystéines.
PCT/US1996/017365 1995-11-02 1996-10-28 Fragments polypeptides derives du produit genique obese WO1997016550A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2000021574A2 (fr) 1998-10-14 2000-04-20 Amgen Inc. Double polyethyleneglycolation de proteines dirigee sur site pour augmenter la bioactivite et la biocompatibilite
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
WO2006096816A3 (fr) * 2005-03-09 2007-05-24 Univ Pittsburgh Polypeptides derives de la leptine humaine et leurs utilisations
WO2010118384A2 (fr) 2009-04-10 2010-10-14 Amylin Pharmaceuticals, Inc. Composés agonistes de l'amyline pour mammifères ayant une carence en oestrogènes
WO2017050793A1 (fr) * 2015-09-22 2017-03-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Polypeptides capables d'inhiber la liaison entre la leptine et la neuropiline-1
US11535659B2 (en) 2010-09-28 2022-12-27 Amryt Pharmaceuticals Inc. Engineered polypeptides having enhanced duration of action

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996005309A2 (fr) * 1994-08-17 1996-02-22 The Rockefeller University Modulateurs de masse corporelle, proteines et acides nucleiques correspondants, et utilisations a des fins therapeutiques et diagnostiques
WO1996023815A1 (fr) * 1995-01-31 1996-08-08 Eli Lilly And Company Anticorps contre les produits du gene ob
WO1996023514A1 (fr) * 1995-01-31 1996-08-08 Eli Lilly And Company Proteines anti-obesite

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996005309A2 (fr) * 1994-08-17 1996-02-22 The Rockefeller University Modulateurs de masse corporelle, proteines et acides nucleiques correspondants, et utilisations a des fins therapeutiques et diagnostiques
WO1996023815A1 (fr) * 1995-01-31 1996-08-08 Eli Lilly And Company Anticorps contre les produits du gene ob
WO1996023514A1 (fr) * 1995-01-31 1996-08-08 Eli Lilly And Company Proteines anti-obesite

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YIYING ZHANG ET AL: "POSITIONAL CLONING OF THE MOUSE OBESE GENE AND ITS HUMAN HOMOLOGUE", NATURE, vol. 372, no. 6505, 1 December 1994 (1994-12-01), pages 425 - 432, XP000602062 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
US7544492B1 (en) 1994-08-17 2009-06-09 The Rockefeller University OB polypeptides, modified forms and derivatives
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
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
US8318666B2 (en) 2001-10-22 2012-11-27 Amgen, Inc. Use of leptin to treat metabolic abnormalities associated with lipoatrophy
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
WO2006096816A3 (fr) * 2005-03-09 2007-05-24 Univ Pittsburgh 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
WO2010118384A2 (fr) 2009-04-10 2010-10-14 Amylin Pharmaceuticals, Inc. Composés agonistes de l'amyline pour mammifères ayant une carence en oestrogènes
US11535659B2 (en) 2010-09-28 2022-12-27 Amryt Pharmaceuticals Inc. Engineered polypeptides having enhanced duration of action
WO2017050793A1 (fr) * 2015-09-22 2017-03-30 INSERM (Institut National de la Santé et de la Recherche Médicale) Polypeptides capables d'inhiber la liaison entre la leptine et la neuropiline-1
US10647752B2 (en) 2015-09-22 2020-05-12 Inserm (Institut National De La Santé Et De La Recherche Medicale) Polypeptides capable of inhibiting the binding between leptin and Neuropilin-1

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