WO1996023514A1 - Anti-obesity proteins - Google Patents

Anti-obesity proteins Download PDF

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Publication number
WO1996023514A1
WO1996023514A1 PCT/US1996/000947 US9600947W WO9623514A1 WO 1996023514 A1 WO1996023514 A1 WO 1996023514A1 US 9600947 W US9600947 W US 9600947W WO 9623514 A1 WO9623514 A1 WO 9623514A1
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WO
WIPO (PCT)
Prior art keywords
xaa
leu
gin
ser
glu
Prior art date
Application number
PCT/US1996/000947
Other languages
French (fr)
Inventor
Margret B. Basinski
Richard D. Dimarchi
David B. Flora
William F. Heath, Jr.
James A. Hoffmann
Brigitte E. Schoner
James E. Shields
David L. Smiley
Original Assignee
Eli Lilly And Company
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
Priority claimed from US08/381,054 external-priority patent/US5569743A/en
Priority claimed from US08/381,666 external-priority patent/US5521283A/en
Priority claimed from US08/381,163 external-priority patent/US5563245A/en
Priority claimed from US08/381,037 external-priority patent/US5563243A/en
Priority claimed from US08/381,034 external-priority patent/US5532336A/en
Priority claimed from US08/381,057 external-priority patent/US5580954A/en
Priority claimed from US08/381,049 external-priority patent/US5574133A/en
Priority claimed from US08/381,050 external-priority patent/US5563244A/en
Priority claimed from US08/381,041 external-priority patent/US5567678A/en
Priority claimed from US08/381,040 external-priority patent/US5552522A/en
Priority claimed from US08/381,370 external-priority patent/US5525705A/en
Priority claimed from US08/383,632 external-priority patent/US5569744A/en
Priority claimed from US08/383,649 external-priority patent/US5567803A/en
Priority claimed from US08/383,650 external-priority patent/US5691309A/en
Priority claimed from US08/384,492 external-priority patent/US5594104A/en
Priority to JP8523609A priority Critical patent/JPH11501297A/en
Application filed by Eli Lilly And Company filed Critical Eli Lilly And Company
Priority to EP96903648A priority patent/EP0836620A1/en
Priority to AU47660/96A priority patent/AU4766096A/en
Publication of WO1996023514A1 publication Critical patent/WO1996023514A1/en

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Classifications

    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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. Most specifically the invention relates to anti-obesity proteins that when administered to a patient regulate fat tissue.
  • the ob I Ob mouse is a model of obesity and diabetes that is known to carry an autosomal recessive trait linked to a mutation in the sixth chromosome. Recently, Yiying Zhang and co-workers published the positional cloning of the mouse gene linked with this condition. Yiying Zhang et al. Nature 372 : 425-32 (1994). This report disclosed a gene coding for a 167 amino acid protein with a 21 amino acid signal peptide that is exclusively expressed in adipose tissue.
  • Physiologist have postulated for years that, when a mammal overeats, the resulting excess fat signals to the brain that the body is obese which, in turn, causes the body to eat less and burn more fuel.
  • the present invention provides biologically active anti-obesity proteins. Such agents therefore allow patients to overcome their obesity handicap and live normal lives with a more normalized risk for type II diabetes, cardiovascular disease and cancer.
  • the present invention is directed to a biologically active anti-obesity proteins of the Formula I:
  • Xaa at position 2 is Gin or Glu
  • Xaa at position 17 is Asn, Asp or Gin;
  • Xaa at position 22 is Thr or Ala
  • Xaa at position 23 is Gin, Glu or absent;
  • Xaa at position 29 is Gin or Glu;
  • Xaa at position 49 is lie, Leu, Met or methionine sulfoxide
  • Xaa at position 51 is Gin or Glu
  • Xaa at position 57 is Gin or Glu
  • Xaa at position 58 is Gin or Glu
  • Xaa at position 63 is lie, Leu, Met or methionine sulfoxide
  • Xaa at position 67 is Asn, Asp or Gin;
  • Xaa at position 70 is Gin or Glu
  • Xaa at position 73 is Asn, Asp or Gin
  • Xaa at position 77 is Asn, Asp or Gin
  • Xaa at position 95 is Trp or Gin
  • Xaa at position 125 is Gin or Glu
  • Xaa at position 129 is Gin or Glu;
  • Xaa at position 131 is lie, Leu, Met or methionine sulfoxide; Xaa at position 133 is Trp or Gin; and Xaa at position 134 is Gin or Glu.
  • the present invention additionally includes fragments of the proteins of Formula I.
  • These proteins are biologically active anti-obesity proteins and are represented by Formulas Ia through In.
  • Formulas Ia through In For clarity purposes, the numbering of the amino acids in Formula I is maintained in Formulas Ia through In. Renumbering the amino acids is unnecessary and would result in confusion.
  • Formula Ia represents amino acids 7 through 146 of SEQ ID NO: 1.
  • the variable cites (Xaa) for each position is the same as previously defined in Formula I unless otherwise specified.
  • Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
  • Xaa at position 49 is lie, Leu, Met, methionine sulfoxide or absent;
  • Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
  • the invention further provides a method of treating obesity, which comprises administering to a mammal in need thereof a protein of any one of Formula I through In.
  • the invention further provides a pharmaceutical formulation, which comprises a protein of any one of Formula I through In together with one or more pharmaceutical acceptable diluents, carriers or excipients therefor.
  • the preferred proteins of the present invention are those wherein:
  • Xaa at position 2 is Gin
  • Xaa at position 17 is Asn
  • Xaa at position 22 is Thr Xaa at position 23 is Gin Xaa at position 29 is Gin Xaa at position 29 is Gin Xaa at position 49 is Met Xaa at position 51 is Gin Xaa at position 57 is Gin Xaa at position 58 is Gin Xaa at position 63 is Met Xaa at position 67 is Asn Xaa at position 70 is Gin Xaa at position 73 is Asn Xaa at position 77 is Asn Xaa at position 95 is Trp Xaa at position 125 is Gin Xaa at position 129 is Gin Xaa at position 131 is Met Xaa at position 133 is Trp Xaa at position 134 is Gin
  • amino acids abbreviations are accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. ⁇ 1.822 (b) (2) (1993) .
  • One skilled in the art would recognize that certain amino acids are prone to rearrangement.
  • Asp may rearrange to aspartimide and isoasparigine as described in I. Sch ⁇ n et al., Int. J. Peotide Protein Res. J .: 485-94 (1979) and references cited therein. These rearrangement derivatives are included within the scope of the present invention.
  • the amino acids are in the L configuration.
  • Base pair (bp) -- refers to DNA or RNA.
  • the abbreviations A,C,G, and T correspond to the 5 ' -monophosphate forms of the nucleotides (deoxy)adenine, (deoxy) cytidine, (deoxy)guanine, and (deoxy) thymine, respectively, when they occur in DNA molecules.
  • the abbreviations U,C,G, and T correspond to the 5 ' -monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules.
  • base pair may refer to a partnership of A with T or C with G.
  • base pair may refer to a partnership of T with U or C with G.
  • EDTA an abbreviation for ethylenediamine tetraacetic acid.
  • ED50 an abbreviation for half-maximal value.
  • FAB-MS an abbreviation for fast atom bombardment mass spectrometry.
  • Immunoreactive Protein(s) a term used to collectively describe antibodies, fragments of antibodies capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived, and single chain polypeptide binding molecules as described in PCT Application No. PCT/US 87/02208, International Publication No. WO 88/01649.
  • mRNA messenger RNA.
  • MWCO an abbreviation for molecular weight cut ⁇ off.
  • Plasmid an extrachromosomal self-replicating genetic element.
  • PMSF an abbreviation for phenylmethylsulfonyl fluoride.
  • Reading frame the nucleotide sequence from which translation occurs "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.
  • Recombinant DNA Cloning Vector 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.
  • Recombinant DNA Expression Vector any recombinant DNA cloning vector in which a promoter has been incorporated.
  • Replicon A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
  • RNA ribonucleic acid.
  • RP-HPLC an abbreviation for reversed-phase high performance liquid chromatography.
  • Transcription the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
  • Translation 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 compound of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.
  • Treating obesity therefor includes the inhibition of food intake, the inhibition of weight gain, and inducing weight loss in patients in need thereof.
  • Vector a replicon used for the transformation of cells in gene manipulation bearing 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 present invention provides biologically active proteins that provide effective treatment for obesity.
  • the proteins are also useful in the production of antibodies for diagnostic use. Many of the claimed proteins offer additional advantages of stability, especially acid stability, and improved absorption characteristics.
  • the claimed proteins ordinarily are prepared by modification of the DNA encoding the claimed protein and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitutional mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis.
  • the mutations that might be made in the DNA encoding the present anti- obesity proteins must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See DeBoer et al. , EP 75,444A (1983) .
  • the compounds of the present invention may be produced either by recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods.
  • the synthesis of the claimed protein may proceed by solid phase peptide synthesis or by recombinant methods.
  • the principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area such as Dugas, H. and Penney, C, Bioor ⁇ anic Chemistry Springer-Verlag, New York, pgs. 54-92 (1981) .
  • peptides may be synthesized by solid-phase methodology utilizing an PE-Applied Biosystems 430A peptide synthesizer (commercially available from Applied Biosystems, Foster City California) and synthesis cycles supplied by Applied Biosystems. Boc amino acids and other reagents are commercially available from PE-Applied Biosystems and other chemical supply houses.
  • Boc deprotection may be accomplished with trifluoroacetic acid (TFA) in methylene chloride.
  • Formyl removal from Trp is accomplished by treatment of the peptidyl resin with 20% piperidine in dimethylformamide for 60 minutes at 4°C.
  • Met (O) can be reduced by treatment of the peptidyl resin with TFA/dimethylsulfide/conHCl (95/5/1) at 25°C for 60 minutes.
  • the peptides may be further deprotected and cleaved from the resin with anhydrous hydrogen fluoride containing a mixture of 10% m-cresol or m- cresol/10% p-thiocresol or m-cresol/p- thiocresol/dimethylsulfide.
  • Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at zero degrees Centigrade or below, preferably -20°C for thirty minutes followed by thirty minutes at 0°C.
  • the peptide/resin is washed with ether.
  • the peptide is extracted with glacial acetic acid and lyophilized. Purification is accomplished by reverse-phase C18 chromatography (Vydac) column in .1% TFA with a gradient of increasing acetonitrile concentration.
  • solid phase synthesis could also be accomplished using the FMOC strategy and a TFA/scavenger cleavage mixture.
  • the claimed proteins may also be produced by recombinant methods. Recombinant methods are preferred if a high yield is desired.
  • the basic steps in the recombinant production of protein include: a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA encoding the claimed protein, b) integrating the coding sequence into an expression vector in a manner suitable for the expression of the protein either alone or as a fusion protein, c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector, and d) recovering and purifying the recombinantly produced protein.
  • the gene encoding the claimed protein may also be created by using polymerase chain reaction (PCR) .
  • the template can be a cDNA library (commercially available from CLONETECH or STRATAGENE) or mRNA isolated from human adipose tissue.
  • PCR polymerase chain reaction
  • cDNA library commercially available from CLONETECH or STRATAGENE
  • mRNA isolated from human adipose tissue Such methodologies are well known in the art Maniatis, sL ⁇ - Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989). ,b, Direct expression or Fusion protein
  • the claimed protein may be made either by direct expression or as fusion protein comprising the claimed protein followed by enzymatic or chemical cleavage.
  • a variety of peptidases e.g. trypsin
  • 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.
  • particular 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. See e.g., Carter P., Site Specific Proteolysis of Fusion Proteins, Ch. 13 in Protein
  • the isolated cDNA coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of this sequence 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 choice of 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 claimed protein.
  • Plasmid pBR322 contains genes for ampicillin and tetracycline resistance 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.
  • 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. 5,304,493, the teachings of which are herein incorporated by reference.
  • the gene encoding A-C-B proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRBl82 with restriction enzymes Ndel and BamHI.
  • the genes encoding the protein of the present invention can be inserted into the plasmid backbone on a Ndel/BamHI restriction fragment cassette.
  • procaryotes are used for cloning of DNA sequences in constructing the vectors useful in the invention.
  • fi ⁇ coli K12 strain 294 ATCC No. 31446
  • Other microbial strains which may be used include £_-. coli B and £ ⁇ coli X1776 (ATCC No. 31537) . These examples are illustrative rather than limiting.
  • Prokaryotes also are used for expression. The aforementioned strains, as well as £--- coli W3110 (prototrophic, ATCC No.
  • Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase (vector pGX2907 [ATCC 39344] contains the replicon and ⁇ -lactamase gene) and lactose promoter systems (Chang ⁇ Jt. a_. , Nature. 275:615 (1978); and Goeddel fit al..
  • alkaline phosphatase alkaline phosphatase
  • the tryptophan (trp) promoter system vector pATHl [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter
  • hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282) .
  • trp tryptophan promoter system
  • vector pATHl ATCC 37695
  • hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282) .
  • other functional bacterial promoters whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the protein using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding protein
  • the protein may be recombinantly produced in eukaryotic expression systems.
  • 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. ⁇ -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, f it al. , Natur . 273 :113 (1978).
  • the entire SV40 genome may be obtained from plasmid pBRSV, ATCC 45019.
  • the immediate early promoter of the human cytomegalovirus may be obtained from plasmid pCMB ⁇ (ATCC
  • promoters from the host cell or related species also are useful herein.
  • Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent having been found 5' (Laimins, L. f it al.. PNAS 78:993 (1981)) and 3' (Lusky, M. L., fit al-. Mol. Cell Bio. 3:1108 (1983)) to the transcription unit, within an intron (Banerji, J. L. f it al. , Cell 12.:729 (1983)) as well as within the coding sequence itself (Osborne, T.
  • enhancer sequences are now known from mammalian genes (globin, RSV, SV40, EMC, elastase, albumin, a-fetoprotein and insulin) .
  • mammalian genes globin, RSV, SV40, EMC, elastase, albumin, a-fetoprotein and insulin
  • an enhancer from a eukaryotic cell virus. Examples include the SV40 late enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • 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.
  • selectable markers for mammalian cells are dihydrofolate reductase (DHFR, which may be derived from the B ⁇ lll/Hindlll restriction fragment of pJOD-10 [ATCC 68815] ) , thymidine kinase (herpes simplex virus thymidine kinase is contained on the BamHI fragment of vP-5 clone [ATCC 2028] ) or neomycin (G418) resistance genes (obtainable from pNN414 yeast artificial chromosome vector [ATCC 37682] ) .
  • DHFR dihydrofolate reductase
  • thymidine kinase herepes simplex virus thymidine kinase is contained on the BamHI fragment of vP-5 clone [ATCC 2028]
  • neomycin (G418) resistance genes obtainable from p
  • the transfected mammalian host cell can survive if placed under selective pressure.
  • selectable markers are successfully transferred into a mammalian host cell
  • the first category is based on a cell's metabolism and the use of a mutant cell line which 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). These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine.
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, Southern P. and Berg, P., J. Molec. APPI. Genet. 1: 327
  • pRc/CMV A preferred vector for eucaryotic 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 £. coli K12 strain DH5a (ATCC 31446) and successful transformants selected by antibiotic resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequence by the method of Messing, ⁇ t al- , Nucleic Acids Res. 1:309 (1981).
  • 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.
  • 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 Maniatis, f it al. , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), or Current Protocols in Molecular Biology
  • Preferred suitable host cells for expressing the vectors encoding the claimed proteins in higher eukaryotes include: African green monkey kidney line cell line transformed by SV40 (COS-7, ATCC CRL-1651); transformed human primary embryonal kidney cell line 293, (Graham, F. L. f it al- > j. Gen viroi. 36:59-72 (1977), virology 12:319-329, virology
  • eukaryotic microbes such as yeast cultures may also be used.
  • Saccharomyces cerevisiae, or common baker's yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available.
  • the plasmid YRp7 for example, (ATCC-40053, Stinchcomb, et al. , Nature 282:39 (1979) ; Kingsman fit al- , £S-Qfi 2:141 (1979);
  • 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, for example ATCC no. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)) .
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (found on plasmid pAPl2BD ATCC 53231 and described in U.S. Patent No. 4,935,350, June 19, 1990) or other glycolytic enzymes such as enolase (found on plasmid pACl ATCC 39532) , glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPCl ATCC 57090, 57091) , zymomonas mobilis (United States Patent No.
  • yeast promoters which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPV ATCC 39475, United States Patent No. 4,840,896) , glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose (GALl found on plasmid pRYl21 ATCC 37658) utilization. Suitable vectors and promoters for use in yeast expression are further described in R.
  • yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae (found in conjunction with the CYCl promoter on plasmid YEpsec--hIlbeta ATCC 67024) , also are advantageously used with yeast promoters.
  • Example 1 A DNA sequence encoding the following protein sequence: Met Arg - SEQ ID NO: 1.
  • a forward primer (5'-GG GG CAT ATG AGG GTA CCT ATC CAG AAA GTC CAG GAT GAC AC) (SEQ ID No: 16) and a reverse primer (5'-GG GG GGATC CTA TTA GCA CCC GGG AGA CAG GTC CAG CTG CCA CAA CAT) (SEQ ID No: 17) is used to amplify sequences from a human fat cell library (commercially available from CLONETECH) .
  • the PCR product is cloned into PCR-Script (available from STRATAGENE) and sequenced.
  • a plasmid containing the DNA sequence encoding the desired claimed protein is constructed to include Ndel and BamHI restriction sites.
  • the plasmid carrying the cloned PCR product is digested with Ndel and BamHI restriction enzymes.
  • the small ⁇ 450bp fragment is gel-purified and ligated into the vector pRBl82 from which the coding sequence for A-C-B proinsulin is deleted.
  • the ligation products are transformed into E. coli DHIOB (commercially available from GIBCO-BRL) and colonies growing on tryptone-yeast (DIFCO) plates supplemented with 10 ⁇ g/mL of tetracycline are analyzed.
  • E. coli DHIOB commercially available from GIBCO-BRL
  • DIFCO tryptone-yeast
  • Plasmid DNA is isolated, digested with Ndel and BamHI and the resulting fragments are separated by agarose gel electrophoresis. Plasmids containing the expected ⁇ 450bp Ndel to BamHI fragment are kept. E. coli B BL21 (DE3) (commercially available from NOVOGEN) are transformed with this second plasmid expression suitable for culture for protein production.
  • E. coli cells used in the preferred practice of the invention as exemplified herein are well known in the art.
  • the precise conditions under which the transformed E ⁇ . coli cells are cultured is dependent on the nature of the £_,_ coli host cell line and the expression or cloning vectors employed.
  • vectors which incorporate thermoinducible promoter-operator regions such as the cl857 thermoinducible lambda-phage promoter-operator region, require a temperature shift from about 30 to about 40 degrees C. in the culture conditions so as to induce protein synthesis.
  • _ thermoinducible promoter-operator regions
  • coli K12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, £. coli K12 L201, L687, L693 , L507, L640, L641, L695, L814 (£. coli B) .
  • the transformed host cells are then 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.
  • Proteins which are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. Kreuger et al., in Protein Folding. Gierasch and King, eds. , pgs 136-142 (1990), American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C. Such protein aggregates must be solubilized to provide further purification and isolation of the desired protein product, Ui.
  • a variety of techniques using strongly denaturing solutions such as guanidinium-HCl and/or weakly denaturing solutions such as dithiothreitol (DTT) are used to solubilize the proteins. Gradual removal of the denaturing agents (often by dialysis) in a solution 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/or the protein in question.
  • Biosynthetic human obese gene product with an intramolecular disulphide was prepared in E. coli , purified and tested as described herein (hereinafter hOB protein) .
  • Human OB protein (19.2 mg) was weighed into a glass vial and dissolved in 4.8 mL phosphate buffered saline, pH 7.4, to give an approximate concentration of 4.0 mg/mL.
  • Complete dissolution of the protein solution was achieved by briefly adjusting the pH to 10.1 with 5N NaOH and then lowering the pH to 7.8 with 5N HCl.
  • the actual concentration of the protein solution was 4.27mg/mL as calculated by UV analysis.
  • the digest was removed from incubation and appeared very turbid with a heavy precipitate at the bottom of the vial.
  • the digestion was terminated by acidification to pH 3.0 with 5N HCl.
  • the digest was centrifuged at 13,600 g for 2 min and the pellet was solubilized with 300ul glacial acetic acid. Upon dilution of the acidified solution with 2.7mL of distilled water some turbidity developed which was removed by centrifugation.
  • the clear supernatant (3mL) was transferred to a glass vial and the remaining gelatinous pellet was solubilized with 150 ⁇ l glacial acetic acid, diluted with an equal volume of distilled water and centrifuged. This supernatant was pooled with the above supernatant and then directly fractionated by RP-HPLC.
  • the fractions were desalted into 70% CH 3 CN/.1% TFA using Waters C 2 Sep-Pak cartridges.
  • the desalted column fractions of the desired peak materials were combined to give a total volume of 4.6mL for each of the peptide sequences from the two semi-preparative RP-HPLC purification runs.
  • the pools were transferred into glass vials in 400 ⁇ l aliquots and lyophilized. Four aliquots of 100 ⁇ l each of the desalted pools were reserved for amino acid and mass spectrometry analysis.
  • Electrospray ionization mass spectrometry of the two desalted pools was performed on a PESciex API III mass spectrometer equipped with a pneumatically-assisted electrospray (Ionspray) interface.
  • Positive ion mass spectra were obtained by continously infusing sample into the interface at a flow rate of 5-20uL/min using a Harvard syringe pump. Data were obtained using an inlet orifice potential of +40V relative to the rod offset potential. Scans were made over a 500-2400u range in 0.lu intervals for a dwell time of 1ms per interval. Multiple scans (3-20) were acquired per sample to provide an averaged final spectrum.
  • the isolated peptide fragments were hydrolyzed under vacuum by a vapor-phase method in a PicoTag Work Station (Waters Associates, Milford, MA) using 6N HCl at 120°C for 21 hours.
  • the hydrolysates were dried down on the Work Station, treated with sample buffer, and analyzed on a Model 6300 Beckman amino acid analyzer.
  • Example 4 A protein of the Formula:
  • hOB protein (lO.Omg) was weighed into a glass vial and dissolved in 2.0ml phosphate buffered saline, pH 7.4. Complete dissolution of the protein solution was achieved by briefly adjusting the pH to 10.0 with 5N NaOH and then lowering the pH to 8.6 with 5N HCl. The actual concentration of solution B was 5.15 mg/ml as calculated by UV analysis.
  • the hOB protein solution B (0.78 ml) was treated with 7M guanidine-hydrochloride (0.86 ml), diluted with phosphate buffered saline (0.36 ml) and adjusted to pH 9.0. The digestion was carried out with lysyl-C endopeptidase
  • Electrospray ionization mass spectrometry of the two desalted pools was performed on a PESciex API III mass spectrometer equipped with a pneumatically-assisted electrospray (Ionspray) interface. Positive ion mass spectra were obtained by continously infusing sample into the interface at a flow rate of 5-20uL/min using a Harvard syringe pump. Data were obtained using an inlet orifice potential of +40V relative to the rod offset potential.
  • the present proteins are expressed as Met-Arg-SEQ ID NO: 1 through 15 so that the expressed proteins may be readily converted to the claimed protein with Cathepsin C.
  • the purification of proteins is by techniques known in the art and includes reverse phase chromatography, affinity chromatography, and size exclusion.
  • the claimed proteins contain two cysteine residues.
  • a di-sulfide bond may be formed to stabilize the protein.
  • the present invention includes proteins of the Formula I through In wherein the Cys at position 91 is crosslinked to Cys at position 141 as well as those proteins without such di-sulfide bonds.
  • the proteins of the present invention may exist, particularly when formulated, as dimers, trimers, tetramers, and other multimers. Such multimers are included within the scope of the present invention.
  • the present invention provides a method for treating obesity. The method comprises administering to the organism an effective amount of anti-obesity protein in a dose between about 1 and 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 0.5 to 100 mg.
  • compounds of the Formula (I) 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 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 instant invention further provides pharmaceutical formulations comprising compounds of the Formula (I through In) .
  • the proteins preferably in the form of a pharmaceutically acceptable salt, can be formulated for nasal, bronchal, transdermal, or parenteral administration for the therapeutic or prophylactic treatment of obesity.
  • compounds of the Formula (I through In) can be admixed with conventional pharmaceutical carriers and excipients.
  • the compositions comprising claimed proteins contain from about 0.1 to 90% by weight of the active protein, preferably in a soluble form, and more generally from about 10 to 30%.
  • the protein is administered in commonly used intravenous fluid (s) and administered by infusion.
  • s intravenous fluid
  • Such fluids for example, physiological saline, Ringer's solution or 5% dextrose solution can be used.
  • a sterile formulation preferably a suitable soluble salt form of a protein of the Formula (I through In), for example the hydrochloride salt
  • a pharmaceutical diluent such as pyrogen-free water (distilled) , physiological saline or 5% glucose solution.
  • a suitable insoluble form of the compound 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.
  • Nasal formulations comprise the protein and carboxyvinyl polymer preferably selected from the group comprising the acrylic acid series hydrophilic crosslinked polymer, e.g. carbopole 934, 940, 941 (Goodrich Co.).
  • the polymer accelerates absorption of the protein, and gives suitable viscosity to prevent discharge from nose.
  • Suitable content of the polymer is 0.05 - 2 weight %.
  • the amount of active compound is commonly 0.1 - 10%.
  • the nasal preparation may be in drop form, spraying applicator or aerosol form.
  • the most closely related biological test is to inject the test article by any of several routes of administration (e.g. i.v., s.c, i.p., or by minipump or cannula) and then to monitor food and water consumption, body weight gain, plasma chemistry or hormones (glucose, insulin, ACTH, corticosterone, GH, T4) over various time periods.
  • routes of administration e.g. i.v., s.c, i.p., or by minipump or cannula
  • Suitable test animals include normal mice (ICR, etc.) and obese mice ( ob/ob , Avy/a, KK-Ay, tubby, fat).
  • the ob/ob mouse model of obesity and diabetes is generally accepted in the art as being indicative of the obesity condition. Controls for non-specific effects for these injections are done using vehicle with or without the active agent of similar composition in the same animal monitoring the same parameters or the active agent itself in animals that are thought to lack the receptor (db/db mice, fa/fa or cp/cp rats) . Proteins demonstrating activity in these models will demonstrate similar activity in other mammals, particularly humans.
  • a similar model is to inject the test article directly into the brain (e.g. i.e.v. injection via lateral or third ventricles, or directly into specific hypothalamic nuclei (e.g. arcuate, paraventricular, perifornical nuclei).
  • the same parameters as above could be measured, or the release of neurotransmitters that are known to regulate feeding or metabolism could be monitored (e.g. NPY, galanin, norepinephrine, dopamine, ⁇ -endorphin release) .
  • 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.

Abstract

The present invention provides anti-obesity proteins, which when administered to a patient regulate fat tissue. Accordingly, such agents allow patients to overcome their obesity handicap and live normal lives with much reduced risk for type II diabetes, cardiovascular disease and cancer.

Description

Anti-obesity proteins
The present invention is in the field of human medicine, particularly in the treatment of obesity and disorders associated with obesity. Most specifically the invention relates to anti-obesity proteins that when administered to a patient regulate fat tissue.
Obesity, and especially upper body obesity, is a common and very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25% of the United States population and 27% of the Canadian population are over weight. Kuczmarski, er. J, Of Clin. Nut, 5_: 495S - 502S (1992); Reeder et. al., Can. Med. Ass. J.. 21: 226-233 (1992). Upper body obesity is the strongest risk factor known for type II diabetes mellitus, and is a strong risk factor for cardiovascular disease and cancer as well. Recent estimates for the medical cost of obesity are $150,000,000,000 world wide. The problem has become serious enough that the surgeon general has begun an initiative to combat the ever increasing adiposity rampant in American society.
Much of this obesity induced pathology can be attributed to the strong association with dyslipidemia, hypertension, and insulin resistance. Many studies have demonstrated that reduction in obesity by diet and exercise reduces these risk factors dramatically. Unfortunately these treatments are largely unsuccessful with a failure rate reaching 95%. This failure may be due to the fact that the condition is strongly associated with genetically inherited factors that contribute to increased appetite, preference for highly caloric foods, reduced physical activity, and increased lipogenic metabolism. This indicates that people inheriting these genetic traits are prone to becoming obese regardless of their efforts to combat the condition. Therefore, a new pharmacological agent that can correct this adiposity handicap and allow the physician to successfully treat obese patients in spite of their genetic inheritance is needed. The ob I Ob mouse is a model of obesity and diabetes that is known to carry an autosomal recessive trait linked to a mutation in the sixth chromosome. Recently, Yiying Zhang and co-workers published the positional cloning of the mouse gene linked with this condition. Yiying Zhang et al. Nature 372 : 425-32 (1994). This report disclosed a gene coding for a 167 amino acid protein with a 21 amino acid signal peptide that is exclusively expressed in adipose tissue.
Physiologist have postulated for years that, when a mammal overeats, the resulting excess fat signals to the brain that the body is obese which, in turn, causes the body to eat less and burn more fuel. G. R. Hervey, Nature 227 :
629-631 (1969) . This "feedback" model is supported by parabiotic experiments, which implicate a circulating hormone controlling adiposity. Based on this model, the protein, which is apparently encoded by the ob gene, is now speculated to be an adiposity regulating hormone. Pharmacological agents which are biologically active and mimic the activity of this protein are useful to help patients regulate their appetite and metabolism and thereby control their adiposity. Until the present invention, such a pharmacological agent was unknown.
The present invention provides biologically active anti-obesity proteins. Such agents therefore allow patients to overcome their obesity handicap and live normal lives with a more normalized risk for type II diabetes, cardiovascular disease and cancer. The present invention is directed to a biologically active anti-obesity proteins of the Formula I:
Formula I: SEQ ID NO: 1
1 5 10 15
Val Xaa Asp Asp Thr Lys Thr Leu lie Lys Thr ie Val Thr Arg
20 25 30 lie Xaa Asp lie Ser His Xaa Xaa Ser Val Ser Ser Lys Xaa Lys
35 40 45
Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro lie Leu Thr 50 55 60
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa lie Leu
65 70 75
Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa Asp Leu
80 85 90
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
95 100 105 Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
110 115 120
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala 125 130 135
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
140 Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 2 is Gin or Glu;
Xaa at position 17 is Asn, Asp or Gin;
Xaa at position 22 is Thr or Ala;
Xaa at position 23 is Gin, Glu or absent; Xaa at position 29 is Gin or Glu;
Xaa at position 49 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 51 is Gin or Glu;
Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu; Xaa at position 63 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 70 is Gin or Glu; Xaa at position 73 is Asn, Asp or Gin; Xaa at position 77 is Asn, Asp or Gin; Xaa at position 95 is Trp or Gin; Xaa at position 125 is Gin or Glu; Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie, Leu, Met or methionine sulfoxide; Xaa at position 133 is Trp or Gin; and Xaa at position 134 is Gin or Glu.
The present invention additionally includes fragments of the proteins of Formula I. These proteins are biologically active anti-obesity proteins and are represented by Formulas Ia through In. For clarity purposes, the numbering of the amino acids in Formula I is maintained in Formulas Ia through In. Renumbering the amino acids is unnecessary and would result in confusion. One of ordinary skill in the art, for example, would appreciate that Formula Ia represents amino acids 7 through 146 of SEQ ID NO: 1. In Formulas Ia through In, the variable cites (Xaa) for each position is the same as previously defined in Formula I unless otherwise specified.
Formula la: SEQ ID NO: 2
10 15 20
Thr Leu lie Lys Thr lie Val Thr Arg lie Xaa Asp lie Ser His
25 30 35
Xaa Xaa Ser Val Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe
40 45 50 lie Pro Gly Leu His Pro lie Leu Thr Leu Ser Lys Xaa Asp Xaa
55 60 65 Thr Leu Ala Val Tyr Xaa Xaa lie Leu Thr Ser Xaa Pro Ser Arg
70 75 80
Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu 85 90 95
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala 100 105 110
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
115 120 125 Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
130 135 140
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
Formula lb: SEQ ID NO: 3
15 20 25
Thr lie Val Thr Arg lie Xaa Asp lie Ser His Xaa Xaa Ser Val 30 35 40
Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe lie Pro Gly Leu
45 50 55
His Pro lie Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val
60 65 70
Tyr Xaa Xaa lie Leu Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa
75 80 85 lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu
90 95 100
Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu 105 110 115
Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser
120 125 130
Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp
135 140
Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
Formula Ic: SEQ ID NO: 4
20 25 30 lie Xaa Asp lie Ser His Xaa Xaa Ser Val Ser Ser Lys Xaa Lys
35 40 45 Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro lie Leu Thr
50 55 60
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa lie Leu 65 70 75
Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa Asp Leu 80 85 90
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
95 100 105
Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
110 115 120
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
125 130 135
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
140 Asp Leu Ser Pro Gly Cys
Formula Id: SEQ ID NO: 5
30 35 40
Xaa Lys Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro lie
45 50 55
Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa
60 65 70 lie Leu Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa
75 80 85
Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser 90 95 100
Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp
105 110 115
Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val
120 125 130
Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa
135 140 Xaa Leu Asp Leu Ser Pro Gly Cys
Formula le: SEQ ID NO: 6
35 40 45 Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro lie Leu Thr
50 55 60
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa lie Leu 65 70 75
Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa Asp Leu 80 85 90
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
95 100 105 Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
110 115 120
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala 125 130 135
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
140 Asp Leu Ser Pro Gly Cys
Formula If: SEQ ID NO: 7
40 45 50 lie Pro Gly Leu His Pro lie Leu Thr Leu Ser Lys Xaa Asp Xaa
55 60 65
Thr Leu Ala Val Tyr Xaa Xaa lie Leu Thr Ser Xaa Pro Ser Arg 70 75 80
Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
85 90 95
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala
100 105 110
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
115 120 125 Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
130 135 140
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
Formula Ig: SEQ ID NO: 8
50 55 60
Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa lie Leu Thr Ser Xaa 65 70 75
Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu
80 85 90
Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu
95 100 105
Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val 110 115 120
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 125 130 135
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser
140 Pro Gly Cys
wherein:
Xaa at position 49 is lie, Leu, Met, methionine sulfoxide or absent;
Formula Ih: SEQ ID NO: 9
60 65 70
Xaa Xaa lie Leu Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie 75 80 85
Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala
90 95 100
Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr
105 110 115
Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr
120 125 130 Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa
135 140
Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
Formula Ii: SEQ ID NO: 10
70 75 80
Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
85 90 95
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala
100 105 110
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
115 120 125
Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
130 135 140 Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys Formula Ij : SEQ ID NO: 11
80 85 90
Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro
95 100 105
Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu
110 115 120 Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu
125 130 135
Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro 140
Gly Cys
Formula Ik: SEQ ID NO: 12
90 95 100
Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu
105 110 115
Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu
120 125 130
Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu
135 140 Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
Formula II: SEQ ID NO: 13
70 75 80 Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu
85 90 95
His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser 100 105 110
Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser
115 120 125
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser
130 135 140
Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys Formula Im: SEQ ID NO: 14
80 85 90
Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu
95 100 105
Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val
110 115 120 Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
125 130 135
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser 140
Pro Gly Cys
Formula In: SEQ ID NO: 15
90 95 100
Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser
105 110 115
Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val
120 125 130
Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa
135 140 Leu Asp Leu Ser Pro Gly Cys
The invention further provides a method of treating obesity, which comprises administering to a mammal in need thereof a protein of any one of Formula I through In. The invention further provides a pharmaceutical formulation, which comprises a protein of any one of Formula I through In together with one or more pharmaceutical acceptable diluents, carriers or excipients therefor.
The preferred proteins of the present invention are those wherein:
Xaa at position 2 is Gin;
Xaa at position 17 is Asn;
Xaa at position 22 is Thr Xaa at position 23 is Gin Xaa at position 29 is Gin Xaa at position 49 is Met Xaa at position 51 is Gin Xaa at position 57 is Gin Xaa at position 58 is Gin Xaa at position 63 is Met Xaa at position 67 is Asn Xaa at position 70 is Gin Xaa at position 73 is Asn Xaa at position 77 is Asn Xaa at position 95 is Trp Xaa at position 125 is Gin Xaa at position 129 is Gin Xaa at position 131 is Met Xaa at position 133 is Trp Xaa at position 134 is Gin
The amino acids abbreviations are accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. § 1.822 (b) (2) (1993) . One skilled in the art would recognize that certain amino acids are prone to rearrangement. For example, Asp may rearrange to aspartimide and isoasparigine as described in I. Schδn et al., Int. J. Peotide Protein Res. J .: 485-94 (1979) and references cited therein. These rearrangement derivatives are included within the scope of the present invention. Unless otherwise indicated the amino acids are in the L configuration.
For purposes of the present invention, as disclosed and claimed herein, the following terms and abbreviations are defined as follows:
Base pair (bp) -- refers to DNA or RNA. The abbreviations A,C,G, and T correspond to the 5 ' -monophosphate forms of the nucleotides (deoxy)adenine, (deoxy) cytidine, (deoxy)guanine, and (deoxy) thymine, respectively, when they occur in DNA molecules. The abbreviations U,C,G, and T correspond to the 5 ' -monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules. In double stranded DNA, base pair may refer to a partnership of A with T or C with G. In a DNA/RNA heteroduplex, base pair may refer to a partnership of T with U or C with G. Chelating Peptide -- An amino acid sequence capable of complexing with a multivalent metal ion.
DNA -- Deoyxribonucleic acid.
EDTA -- an abbreviation for ethylenediamine tetraacetic acid. ED50 -- an abbreviation for half-maximal value.
FAB-MS -- an abbreviation for fast atom bombardment mass spectrometry.
Immunoreactive Protein(s) -- a term used to collectively describe antibodies, fragments of antibodies capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived, and single chain polypeptide binding molecules as described in PCT Application No. PCT/US 87/02208, International Publication No. WO 88/01649. mRNA -- messenger RNA.
MWCO -- an abbreviation for molecular weight cut¬ off.
Plasmid -- an extrachromosomal self-replicating genetic element. PMSF -- an abbreviation for phenylmethylsulfonyl fluoride.
Reading frame -- the nucleotide sequence from which translation occurs "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. In the creation of fusion proteins containing a chelating peptide, the reading frame of the DNA sequence encoding the structural protein must be maintained in the DNA sequence encoding the chelating peptide. Recombinant DNA Cloning Vector -- 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.
Recombinant DNA Expression Vector -- any recombinant DNA cloning vector in which a promoter has been incorporated.
Replicon -- A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector. RNA -- ribonucleic acid. RP-HPLC -- an abbreviation for reversed-phase high performance liquid chromatography.
Transcription -- the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence. Translation -- the process whereby the genetic information of messenger RNA is used to specify and direct the synthesis of a polypeptide chain.
Tris -- an abbreviation for tris(hydroxymethyl) - aminomethane. 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 compound of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating obesity therefor includes the inhibition of food intake, the inhibition of weight gain, and inducing weight loss in patients in need thereof.
Vector -- a replicon used for the transformation of cells in gene manipulation bearing 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.
X-gal -- an abbreviation for 5-bromo-4-chloro-3- idolyl beta-D-galactoside.
Yiying Zhang et al. in Nature 372: 425-32 (December
1994) report the cloning of the murine obese { ob) mouse gene and present mouse DNA and the naturally occurring amino acid sequence of the obesity protein for the mouse and human. This protein is speculated to be a hormone that is secreted by fat cells and controls body weight.
The present invention provides biologically active proteins that provide effective treatment for obesity. The proteins are also useful in the production of antibodies for diagnostic use. Many of the claimed proteins offer additional advantages of stability, especially acid stability, and improved absorption characteristics.
The claimed proteins ordinarily are prepared by modification of the DNA encoding the claimed protein and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitutional mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis. The mutations that might be made in the DNA encoding the present anti- obesity proteins must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See DeBoer et al. , EP 75,444A (1983) .
The compounds of the present invention may be produced either by recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods.
£ > Solid Phase The synthesis of the claimed protein may proceed by solid phase peptide synthesis or by recombinant methods. The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts in the area such as Dugas, H. and Penney, C, Bioorσanic Chemistry Springer-Verlag, New York, pgs. 54-92 (1981) . For example, peptides may be synthesized by solid-phase methodology utilizing an PE-Applied Biosystems 430A peptide synthesizer (commercially available from Applied Biosystems, Foster City California) and synthesis cycles supplied by Applied Biosystems. Boc amino acids and other reagents are commercially available from PE-Applied Biosystems and other chemical supply houses. Sequential Boc chemistry using double couple protocols are applied to the starting p-methyl benzhydryl amine resins for the production of C-terminal carboxamides. For the production of C-terminal acids, the corresponding PAM resin is used. Arginine, Asparagine, Glutamine, Histidine and Methionine are coupled using preformed hydroxy benzotriazole esters. The following side chain protection may be used: Arg, Tosyl
Asp, cyclohexyl or benzyl Cys, 4-methylbenzyl Glu, cyclohexyl His, benzyloxymethyl Lys, 2-chlorobenzyloxycarbonyl
Met, sulfoxide Ser, Benzyl Thr, Benzyl Trp, formyl Tyr, 4-bromo carbobenzoxy
Boc deprotection may be accomplished with trifluoroacetic acid (TFA) in methylene chloride. Formyl removal from Trp is accomplished by treatment of the peptidyl resin with 20% piperidine in dimethylformamide for 60 minutes at 4°C. Met (O) can be reduced by treatment of the peptidyl resin with TFA/dimethylsulfide/conHCl (95/5/1) at 25°C for 60 minutes. Following the above pre-treatments, the peptides may be further deprotected and cleaved from the resin with anhydrous hydrogen fluoride containing a mixture of 10% m-cresol or m- cresol/10% p-thiocresol or m-cresol/p- thiocresol/dimethylsulfide. Cleavage of the side chain protecting group(s) and of the peptide from the resin is carried out at zero degrees Centigrade or below, preferably -20°C for thirty minutes followed by thirty minutes at 0°C. After removal of the HF, the peptide/resin is washed with ether. The peptide is extracted with glacial acetic acid and lyophilized. Purification is accomplished by reverse-phase C18 chromatography (Vydac) column in .1% TFA with a gradient of increasing acetonitrile concentration.
One skilled in the art recognizes that the solid phase synthesis could also be accomplished using the FMOC strategy and a TFA/scavenger cleavage mixture.
EJ Recombinant synthesis
The claimed proteins may also be produced by recombinant methods. Recombinant methods are preferred if a high yield is desired. The basic steps in the recombinant production of protein include: a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA encoding the claimed protein, b) integrating the coding sequence into an expression vector in a manner suitable for the expression of the protein either alone or as a fusion protein, c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector, and d) recovering and purifying the recombinantly produced protein.
2.a. Gene Construction Synthetic genes, the j vitro or jj} vivo transcription and translation of which will result in the production of the protein may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed which encode the claimed proteins. In the preferred practice of the invention, synthesis is achieved by recombinant DNA technology.
Methodology of synthetic gene construction is well known in the art. For example, see Brown, ≤£. a_l. (1979) Methods in Enzymology, Academic Press, N.Y., Vol. β_, pgs. 109-151. The DNA sequence corresponding to the synthetic claimed protein gene may be generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404) .
It may desirable in some applications to modify the coding sequence of the claimed protein so as to incorporate a convenient protease sensitive cleavage site, e.g., between the signal peptide and the structural protein facilitating the controlled excision of the signal peptide from the fusion protein construct.
The gene encoding the claimed protein may also be created by using polymerase chain reaction (PCR) . The template can be a cDNA library (commercially available from CLONETECH or STRATAGENE) or mRNA isolated from human adipose tissue. Such methodologies are well known in the art Maniatis, sL ≤ - Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989). ,b, Direct expression or Fusion protein
The claimed protein may be made either by direct expression or as fusion protein comprising the claimed protein followed by enzymatic or chemical cleavage. A variety of peptidases (e.g. trypsin) 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. 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. See e.g., Carter P., Site Specific Proteolysis of Fusion Proteins, Ch. 13 in Protein
Purification: From Molecular Mechanisms to Larαe Scale Processes. American Chemical Soc, Washington, D.C. (1990) .
2.C. Vector Construction Construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to form the plasmids required. To effect the translation of the 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. The claimed protein is a relatively large protein. A synthetic coding sequence is 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. The isolated cDNA coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of this sequence 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 choice of 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 claimed protein.
In general, plasmid vectors containing promoters and control sequences which are derived from species compatible with the host cell are used with these hosts. The vector ordinarily carries a replication site as well as marker sequences which are capable of providing phenotypic selection in transformed cells. For example, £_.. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar, ≤-t _l. , Gene 2: 95 (1977)). Plasmid pBR322 contains genes for ampicillin and tetracycline resistance 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.
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. 5,304,493, the teachings of which are herein incorporated by reference. The gene encoding A-C-B proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRBl82 with restriction enzymes Ndel and BamHI. The genes encoding the protein of the present invention can be inserted into the plasmid backbone on a Ndel/BamHI restriction fragment cassette.
2.d. Proπarvotic expression
In general, procaryotes are used for cloning of DNA sequences in constructing the vectors useful in the invention. For example, fi^ coli K12 strain 294 (ATCC No. 31446) is particularly useful. Other microbial strains which may be used include £_-. coli B and £^ coli X1776 (ATCC No. 31537) . These examples are illustrative rather than limiting. Prokaryotes also are used for expression. The aforementioned strains, as well as £--- coli W3110 (prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, and various pseudomonas species may be used. Promoters suitable for use with prokaryotic hosts include the β-lactamase (vector pGX2907 [ATCC 39344] contains the replicon and β-lactamase gene) and lactose promoter systems (Chang ≤Jt. a_. , Nature. 275:615 (1978); and Goeddel fit al.. _i__ -2fll:544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] 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 (isolatable from plasmid pDR540 ATCC-37282) . However, other functional bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the protein using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding protein.
2-e. Eucarvotic expression
The protein may be recombinantly produced in eukaryotic expression systems. 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. β-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, fit al. , Natur . 273 :113 (1978). The entire SV40 genome may be obtained from plasmid pBRSV, ATCC 45019. The immediate early promoter of the human cytomegalovirus may be obtained from plasmid pCMBβ (ATCC
77177). Of course, promoters from the host cell or related species also are useful herein.
Transcription of a DNA encoding the claimed protein by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent having been found 5' (Laimins, L. fit al.. PNAS 78:993 (1981)) and 3' (Lusky, M. L., fit al-. Mol. Cell Bio. 3:1108 (1983)) to the transcription unit, within an intron (Banerji, J. L. fit al. , Cell 12.:729 (1983)) as well as within the coding sequence itself (Osborne, T. F., fit al., Mol. Cell Bio. 4:1293 (1984)). Many enhancer sequences are now known from mammalian genes (globin, RSV, SV40, EMC, elastase, albumin, a-fetoprotein and insulin) . Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 late enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) 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 Bσlll/Hindlll restriction fragment of pJOD-10 [ATCC 68815] ) , thymidine kinase (herpes simplex virus thymidine kinase is contained on the BamHI fragment of vP-5 clone [ATCC 2028] ) or neomycin (G418) resistance genes (obtainable from pNN414 yeast artificial chromosome vector [ATCC 37682] ) . When such selectable markers are successfully transferred into a mammalian host cell, the transfected mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which 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). These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. 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 nonsupplemented media. The second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, Southern P. and Berg, P., J. Molec. APPI. Genet. 1: 327
(1982), mycophenolic acid, Mulligan, R. C. and Berg, P. Science 209:1422 (1980), or hygromycin, Sugden, B. et al. , Mol Cell. Biol. 5:410-413 (1985). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin) , xgpt (mycophenolic acid) or hygromycin, respectively.
A preferred vector for eucaryotic expression is pRc/CMV. pRc/CMV is commercially available from Invitrogen Corporation, 3985 Sorrento Valley Blvd., San Diego, CA 92121.
To confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform £. coli K12 strain DH5a (ATCC 31446) and successful transformants selected by antibiotic resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequence by the method of Messing, ≤t al- , Nucleic Acids Res. 1:309 (1981).
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. 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 Maniatis, fit al. , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), or Current Protocols in Molecular Biology
(1989) and supplements.
Preferred suitable host cells for expressing the vectors encoding the claimed proteins in higher eukaryotes include: African green monkey kidney line cell line transformed by SV40 (COS-7, ATCC CRL-1651); transformed human primary embryonal kidney cell line 293, (Graham, F. L. fit al- > j. Gen viroi. 36:59-72 (1977), virology 12:319-329, virology
££.:10-21) ; baby hamster kidney cells (BHK-21(C-13) , ATCC CCL- 10, Virology 16_:147 (1962)); Chinese hamster ovary cells CHO- DHFR" (ATCC CRL-9096), mouse Sertoli cells (TM4, ATCC CRL- 1715, Biol. Reorod. 23:243-250 (1980)); african green monkey kidney cells (VERO 76, ATCC CRL-1587); human cervical epitheloid 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) .
2,f . Yeast expression
In addition to prokaryotes, eukaryotic microbes such as yeast cultures may also be used. Saccharomyces cerevisiae, or common baker's yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available. For expression in Saccharomyces, the plasmid YRp7, for example, (ATCC-40053, Stinchcomb, et al. , Nature 282:39 (1979) ; Kingsman fit al- , £S-Qfi 2:141 (1979);
Tschemper fit al- , Gene 1£:157 (1980)) is commonly used. 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, for example ATCC no. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)) .
Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (found on plasmid pAPl2BD ATCC 53231 and described in U.S. Patent No. 4,935,350, June 19, 1990) or other glycolytic enzymes such as enolase (found on plasmid pACl ATCC 39532) , glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPCl ATCC 57090, 57091) , zymomonas mobilis (United States Patent No. 5,000,000 issued March 19, 1991), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPV ATCC 39475, United States Patent No. 4,840,896) , glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose (GALl found on plasmid pRYl21 ATCC 37658) utilization. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman fit al- • European Patent Publication No. 73,657A. Yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae (found in conjunction with the CYCl promoter on plasmid YEpsec--hIlbeta ATCC 67024) , also are advantageously used with yeast promoters.
The following examples are presented to further illustrate the preparation of the claimed proteins. The scope of the present invention is not to be construed as merely consisting of the following examples.
Example 1 A DNA sequence encoding the following protein sequence: Met Arg - SEQ ID NO: 1.
is obtained using standard PCR methodology. A forward primer (5'-GG GG CAT ATG AGG GTA CCT ATC CAG AAA GTC CAG GAT GAC AC) (SEQ ID No: 16) and a reverse primer (5'-GG GG GGATC CTA TTA GCA CCC GGG AGA CAG GTC CAG CTG CCA CAA CAT) (SEQ ID No: 17) is used to amplify sequences from a human fat cell library (commercially available from CLONETECH) . The PCR product is cloned into PCR-Script (available from STRATAGENE) and sequenced.
Example 2 Vector Construction
A plasmid containing the DNA sequence encoding the desired claimed protein is constructed to include Ndel and BamHI restriction sites. The plasmid carrying the cloned PCR product is digested with Ndel and BamHI restriction enzymes. The small ~ 450bp fragment is gel-purified and ligated into the vector pRBl82 from which the coding sequence for A-C-B proinsulin is deleted. The ligation products are transformed into E. coli DHIOB (commercially available from GIBCO-BRL) and colonies growing on tryptone-yeast (DIFCO) plates supplemented with 10 μg/mL of tetracycline are analyzed. Plasmid DNA is isolated, digested with Ndel and BamHI and the resulting fragments are separated by agarose gel electrophoresis. Plasmids containing the expected ~ 450bp Ndel to BamHI fragment are kept. E. coli B BL21 (DE3) (commercially available from NOVOGEN) are transformed with this second plasmid expression suitable for culture for protein production.
The techniques of transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as Maniatis, et al. (1988) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor
Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York or Current Protocols in Molecular Biology (1989) and supplements. The techniques involved in the transformation of E. coli cells used in the preferred practice of the invention as exemplified herein are well known in the art. The precise conditions under which the transformed E^. coli cells are cultured is dependent on the nature of the £_,_ coli host cell line and the expression or cloning vectors employed. For example, vectors which incorporate thermoinducible promoter-operator regions, such as the cl857 thermoinducible lambda-phage promoter-operator region, require a temperature shift from about 30 to about 40 degrees C. in the culture conditions so as to induce protein synthesis. In the preferred embodiment of the invention _. coli K12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, £. coli K12 L201, L687, L693 , L507, L640, L641, L695, L814 (£. coli B) . The transformed host cells are then 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.
Proteins which are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. Kreuger et al., in Protein Folding. Gierasch and King, eds. , pgs 136-142 (1990), American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C. Such protein aggregates must be solubilized to provide further purification and isolation of the desired protein product, Ui. A variety of techniques using strongly denaturing solutions such as guanidinium-HCl and/or weakly denaturing solutions such as dithiothreitol (DTT) are used to solubilize the proteins. Gradual removal of the denaturing agents (often by dialysis) in a solution 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/or the protein in question.
Example 3
A protein of the Formula:
90 95 100
Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser
105 110 115
Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val
120 125 130 Ala Leu Ser Arg Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin
135 140
Leu Asp Leu Ser Pro Gly Cys
was prepared as follows.
Biosynthetic human obese gene product with an intramolecular disulphide was prepared in E. coli , purified and tested as described herein (hereinafter hOB protein) . Human OB protein (19.2 mg) was weighed into a glass vial and dissolved in 4.8 mL phosphate buffered saline, pH 7.4, to give an approximate concentration of 4.0 mg/mL. Complete dissolution of the protein solution was achieved by briefly adjusting the pH to 10.1 with 5N NaOH and then lowering the pH to 7.8 with 5N HCl. The actual concentration of the protein solution was 4.27mg/mL as calculated by UV analysis. The hOB protein solution (4.7mL) was digested with lysyl-C endopeptidase (E:S=1:500 wt/wt) for 2 hrs. at 37°C. The digest was removed from incubation and appeared very turbid with a heavy precipitate at the bottom of the vial. The digestion was terminated by acidification to pH 3.0 with 5N HCl. The digest was centrifuged at 13,600 g for 2 min and the pellet was solubilized with 300ul glacial acetic acid. Upon dilution of the acidified solution with 2.7mL of distilled water some turbidity developed which was removed by centrifugation. The clear supernatant (3mL) was transferred to a glass vial and the remaining gelatinous pellet was solubilized with 150μl glacial acetic acid, diluted with an equal volume of distilled water and centrifuged. This supernatant was pooled with the above supernatant and then directly fractionated by RP-HPLC.
The digest fragments were fractionated using a RP-HPLC system consisting of Beckman 110A pumps and a Pharmacia detector. Purification of the lysyl-C endopeptidase peptides was performed on a C4~Vydac column (10 x 250 mm) with a gradient composed of buffer A (2 parts CH3CN/98 parts 0.05M Na2S0 , pH 2.3) and buffer B (70 parts CH3CN/30 parts 0.05M Na2S04, pH 2.3). The peptides were eluted at a flow rate of 2 mL/min with a linear gradient of 20 to 80% buffer B over 180 min. The column effluent was monitored at 214 nm, R=1.0 AUFS and collected manually. The desired peak materials eluted at about 42.1% CH3CN and 48.2% CH3CN for the peptide fragments
54-95 and 95-146, respectively. The fractions were desalted into 70% CH3CN/.1% TFA using Waters C2 Sep-Pak cartridges. In order to accumulate sufficient amounts of desired peptide sequence materials the lysyl-C endopeptidase digest and the RP-HPLC purification process were repeated exactly as described. The desalted column fractions of the desired peak materials were combined to give a total volume of 4.6mL for each of the peptide sequences from the two semi-preparative RP-HPLC purification runs. The pools were transferred into glass vials in 400 μl aliquots and lyophilized. Four aliquots of 100 μl each of the desalted pools were reserved for amino acid and mass spectrometry analysis.
Electrospray ionization mass spectrometry of the two desalted pools was performed on a PESciex API III mass spectrometer equipped with a pneumatically-assisted electrospray (Ionspray) interface. Positive ion mass spectra were obtained by continously infusing sample into the interface at a flow rate of 5-20uL/min using a Harvard syringe pump. Data were obtained using an inlet orifice potential of +40V relative to the rod offset potential. Scans were made over a 500-2400u range in 0.lu intervals for a dwell time of 1ms per interval. Multiple scans (3-20) were acquired per sample to provide an averaged final spectrum. The isolated peptide fragments were hydrolyzed under vacuum by a vapor-phase method in a PicoTag Work Station (Waters Associates, Milford, MA) using 6N HCl at 120°C for 21 hours. The hydrolysates were dried down on the Work Station, treated with sample buffer, and analyzed on a Model 6300 Beckman amino acid analyzer. The results of the mass spectrometry MW observed 5489.9 (MW theoretical: 5490.2). The results were also confirmed by amino acid analysis.
Example 4 A protein of the Formula:
50 55 60
Met Asp Gin Thr Leu Ala Val Tyr Gin Gin lie Leu Thr Ser Met
65 70 75
Pro Ser Arg Asn Val lie Gin lie Ser Asn Asp Leu Glu Asn Leu
80 85 90
Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu
95 100 105 Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val 110 115 120
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
125 130 135 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser
140 Pro Gly Cys
was prepared as follows. hOB protein (lO.Omg) was weighed into a glass vial and dissolved in 2.0ml phosphate buffered saline, pH 7.4. Complete dissolution of the protein solution was achieved by briefly adjusting the pH to 10.0 with 5N NaOH and then lowering the pH to 8.6 with 5N HCl. The actual concentration of solution B was 5.15 mg/ml as calculated by UV analysis.
The hOB protein solution B (0.78 ml) was treated with 7M guanidine-hydrochloride (0.86 ml), diluted with phosphate buffered saline (0.36 ml) and adjusted to pH 9.0. The digestion was carried out with lysyl-C endopeptidase
(E:S=1:1000 wt/wt) for 30 min. at 37°C. The clear solution was removed from the incubator and acidified to pH 2.0 with glacial acetic acid (0.15ml) to terminate the digest. The acidified solution was directly fractionated by RP-HPLC. The digest fragments were fractionated using a RP-HPLC system consisting of Beckman 110A pumps and a Pharmacia detector. Purification of the lysyl-C endopeptidase peptides was performed on a C4~Vydac column (10 x 250mm) with a gradient composed of buffer A (2 parts CH3CN/98 parts 0.05M Na2S0 , pH 2.3) and buffer B (70 parts CH3CN/30 parts 0.05M a2S04, pH 2.3). The peptides were eluted at a flow rate of 2ml/min with a linear gradient of 20 to 80% buffer B over 180 min. The column effluent was monitored at 214nm, R=1.0 AUFS and collected manually. The desired peak materials eluted at about 42.1, 47.6, and 49.3% CH3CN for the peptide fragments
54-94, 95-146, and 54-146, respectively. The fractions were desalted into 70% CH3CN/.1% TFA using Waters C2 Sep-Pak cartridges. Electrospray ionization mass spectrometry of the two desalted pools was performed on a PESciex API III mass spectrometer equipped with a pneumatically-assisted electrospray (Ionspray) interface. Positive ion mass spectra were obtained by continously infusing sample into the interface at a flow rate of 5-20uL/min using a Harvard syringe pump. Data were obtained using an inlet orifice potential of +40V relative to the rod offset potential. Scans were made over a 500-2400u range in O.lu intervals for a dwell time of 1ms per interval. Multiple scans (3-20) were acquired per sample to provide an averaged final spectrum. The results of the mass spectrometry were observed MW 10,188.4 (theoretical 10,188.7).
Preferably, the present proteins are expressed as Met-Arg-SEQ ID NO: 1 through 15 so that the expressed proteins may be readily converted to the claimed protein with Cathepsin C. The purification of proteins is by techniques known in the art and includes reverse phase chromatography, affinity chromatography, and size exclusion. The claimed proteins contain two cysteine residues.
Thus, a di-sulfide bond may be formed to stabilize the protein. The present invention includes proteins of the Formula I through In wherein the Cys at position 91 is crosslinked to Cys at position 141 as well as those proteins without such di-sulfide bonds.
In addition the proteins of the present invention may exist, particularly when formulated, as dimers, trimers, tetramers, and other multimers. Such multimers are included within the scope of the present invention. The present invention provides a method for treating obesity. The method comprises administering to the organism an effective amount of anti-obesity protein in a dose between about 1 and 1000 μg/kg. A preferred dose is from about 10 to 100 μg/kg of active compound. A typical daily dose for an adult human is from about 0.5 to 100 mg. In practicing this method, compounds of the Formula (I) 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 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 instant invention further provides pharmaceutical formulations comprising compounds of the Formula (I through In) . The proteins, preferably in the form of a pharmaceutically acceptable salt, can be formulated for nasal, bronchal, transdermal, or parenteral administration for the therapeutic or prophylactic treatment of obesity. For example, compounds of the Formula (I through In) can be admixed with conventional pharmaceutical carriers and excipients. The compositions comprising claimed proteins contain from about 0.1 to 90% by weight of the active protein, preferably in a soluble form, and more generally from about 10 to 30%.
For intravenous (IV) use, 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.
For intramuscular preparations, a sterile formulation, preferably a suitable soluble salt form of a protein of the Formula (I through In), for example the hydrochloride salt, can be dissolved and administered in a pharmaceutical diluent such as pyrogen-free water (distilled) , physiological saline or 5% glucose solution. A suitable insoluble form of the compound 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.
It may also be desirable to administer the compounds of Formula (I through In) intranasally.
Formulations useful in the intranasal absorption of proteins are well known in the art. Nasal formulations comprise the protein and carboxyvinyl polymer preferably selected from the group comprising the acrylic acid series hydrophilic crosslinked polymer, e.g. carbopole 934, 940, 941 (Goodrich Co.). The polymer accelerates absorption of the protein, and gives suitable viscosity to prevent discharge from nose. Suitable content of the polymer is 0.05 - 2 weight %. By neutralisation of the polymer with basic substance, thickening effect is increased. The amount of active compound is commonly 0.1 - 10%. The nasal preparation may be in drop form, spraying applicator or aerosol form.
The ability of the present compounds to treat obesity is demonstrated in vivo as follows:
Biological Testing for Anti-obesitv proteins
Parabiotic experiments suggest that a 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. Therefore, the most closely related biological test is to inject the test article by any of several routes of administration (e.g. i.v., s.c, i.p., or by minipump or cannula) and then to monitor food and water consumption, body weight gain, plasma chemistry or hormones (glucose, insulin, ACTH, corticosterone, GH, T4) over various time periods.
Suitable test animals include normal mice (ICR, etc.) and obese mice ( ob/ob , Avy/a, KK-Ay, tubby, fat). The ob/ob mouse model of obesity and diabetes is generally accepted in the art as being indicative of the obesity condition. Controls for non-specific effects for these injections are done using vehicle with or without the active agent of similar composition in the same animal monitoring the same parameters or the active agent itself in animals that are thought to lack the receptor (db/db mice, fa/fa or cp/cp rats) . Proteins demonstrating activity in these models will demonstrate similar activity in other mammals, particularly humans. Since the target tissue is expected to be the hypothalamus where food intake and lipogenic state are regulated, a similar model is to inject the test article directly into the brain (e.g. i.e.v. injection via lateral or third ventricles, or directly into specific hypothalamic nuclei (e.g. arcuate, paraventricular, perifornical nuclei). The same parameters as above could be measured, or the release of neurotransmitters that are known to regulate feeding or metabolism could be monitored (e.g. NPY, galanin, norepinephrine, dopamine, β-endorphin release) .
Similar studies are accomplished in vitro using isolated hypothalamic tissue in a perifusion or tissue bath system. In this situation, the release of neurotransmitters or electrophysiological changes is monitored.
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. However, 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. Furthermore, 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.

Claims

We claim :
1. A protein of the formula:
1 5 10 15 Val Xaa Asp Asp Thr Lys Thr Leu lie Lys Thr lie Val Thr Arg
20 25 30 lie Xaa Asp lie Ser His Xaa Xaa Ser Val Ser Ser Lys Xaa Lys 35 40 45
Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro lie Leu Thr
50 55 60
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa lie Leu
65 70 75
Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa Asp Leu
80 85 90 Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
95 100 105
Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu 110 115 120
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
125 130 135
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
140 Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 2 is Gin or Glu; Xaa at position 17 is Asn, Asp or Gin;
Xaa at position 22 is Thr or Ala;
Xaa at position 23 is Gin, Glu or absent;
Xaa at position 29 is Gin or Glu;
Xaa at position 49 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 51 is Gin or Glu;
Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu;
Xaa at position 63 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 70 is Gin or Glu; Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu; Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
2. A protein of the formula:
10 15 20 Thr Leu lie Lys Thr lie Val Thr Arg lie Xaa Asp lie Ser His
25 30 35
Xaa Xaa Ser Val Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe 40 45 50 lie Pro Gly Leu His Pro lie Leu Thr Leu Ser Lys Xaa Asp Xaa
55 60 65
Thr Leu Ala Val Tyr Xaa Xaa lie Leu Thr Ser Xaa Pro Ser Arg
70 75 80
Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
85 90 95 Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala
100 105 110
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala 115 120 125
Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
130 135 140
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys wherein:
Xaa at position 17 is Asn, Asp or Gin;
Xaa at position 22 is Thr or Ala;
Xaa at position 23 is Gin, Glu or absent;
Xaa at position 29 is Gin or Glu; Xaa at position 49 is lie. Leu, Met or methionine sulfoxide;
Xaa at position 51 is Gin or Glu;
Xaa at position 57 is Gin or Glu; Xaa at position 58 is Gin or Glu;
Xaa at position 63 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 70 is Gin or Glu; Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu; Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
3. A protein of the formula:
15 20 25
Thr lie Val Thr Arg lie Xaa Asp lie Ser His Xaa Xaa Ser Val
30 35 40
Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe lie Pro Gly Leu
45 50 55 His Pro lie Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val
60 65 70
Tyr Xaa Xaa lie Leu Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa 75 80 85 lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu
90 95 100
Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu
105 110 115
Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser
120 125 130 Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp 135 140
Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 17 is Asn, Asp or Gin;
Xaa at position 22 is Thr or Ala;
Xaa at position 23 is Gin, Glu or absent;
Xaa at position 29 is Gin or Glu; Xaa at position 49 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 51 is Gin or Glu;
Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu; Xaa at position 63 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin; Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
4. A protein of the formula:
20 25 30 lie Xaa Asp lie Ser His Xaa Xaa Ser Val Ser Ser Lys Xaa Lys 35 40 45
Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro lie Leu Thr
50 55 60
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa lie Leu
65 70 75
Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa Asp Leu 80 85 90
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
95 100 105
Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
110 115 120
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
125 130 135
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
140 Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 17 is Asn, Asp or Gin;
Xaa at position 22 is Thr or Ala; Xaa at position 23 is Gin, Glu or absent;
Xaa at position 29 is Gin or Glu;
Xaa at position 49 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 51 is Gin or Glu; Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu;
Xaa at position 63 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gin; Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu; Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof. 5. A protein of the formula:
30 35 40
Xaa Lys Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro lie
45 50 55
Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa
60 65 70 lie Leu Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa
75 80 85
Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser 90 95 100
Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp
105 110 115
Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val
120 125 130
Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa
135 140 Xaa Leu Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 29 is Gin or Glu;
Xaa at position 49 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 51 is Gin or Glu;
Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu;
Xaa at position 63 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin; Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu; Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
6. A protein of the formula:
35 40 45 Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro lie Leu Thr
50 55 60
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa lie Leu 65 70 75
Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa Asp Leu
80 85 90
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
95 100 105
Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
110 115 120 Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
125 130 135
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu 140
Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 49 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 51 is Gin or Glu;
Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu;
Xaa at position 63 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin; Xaa at position 95 is Trp or Gin; Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie, Leu, Met or methionine sulfoxide; Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
7. A protein of the formula:
40 45 50 lie Pro Gly Leu His Pro lie Leu Thr Leu Ser Lys Xaa Asp Xaa
55 60 65 Thr Leu Ala Val Tyr Xaa Xaa lie Leu Thr Ser Xaa Pro Ser Arg
70 75 80
Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu 85 90 95
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala
100 105 110
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
115 120 125
Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
130 135 140 Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 49 is lie, Leu, Met or methionine sulfoxide; Xaa at position 51 is Gin or Glu;
Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu;
Xaa at position 63 is lie, Leu, Met or methionine sulfoxide; Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin; Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie, Leu, Met or methionine sulfoxide; Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
8. A protein of the formula:
50 55 60
Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa lie Leu Thr Ser Xaa
65 70 75 Pro Ser Arg Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu
80 85 90
Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu 95 100 105
Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val
110 115 120
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
125 130 135
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser
140 Pro Gly Cys
wherein:
Xaa at position 49 is lie, Leu, Met, methionine sulfoxide or absent; Xaa at position 51 is Gin or Glu;
Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu;
Xaa at position 63 is lie, Leu, Met or methionine sulfoxide; Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin; Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie. Leu, Met or methionine sulfoxide; Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
9. A protein of the formula:
60 65 70
Xaa Xaa lie Leu Thr Ser Xaa Pro Ser Arg Xaa Val lie Xaa lie
75 80 85 Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala
90 95 100
Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr
105 110 115
Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr
120 125 130
Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa
135 140
Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
wherein: Xaa at position 57 is Gin or Glu;
Xaa at position 58 is Gin or Glu;
Xaa at position 63 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gin; Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu; Xaa at position 129 is Gin or Glu; Xaa at position 131 is lie. Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
10. A protein of the formula:
70 75 80 Xaa Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
85 90 95
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala 100 105 110
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
115 120 125
Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
130 135 140
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
wherein: Xaa at position 67 is Asn, Asp or Gin;
Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin; Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
11. A protein of the formula:
80 85 90
Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro
95 100 105
Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu
110 115 120 Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu
125 130 135
Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro 140
Gly Cys
wherein:
Xaa at position 95 is Trp or Gin; Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
12. A protein of the formula:
90 95 100
Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu
105 110 115
Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu
120 125 130
Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu
135 140 Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 95 is Trp or Gin; Xaa at position 125 is Gin or Glu; Xaa at position 129 is Gin or Glu; Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
13. A protein of the formula:
70 75 80 Val lie Xaa lie Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu
85 90 95
His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser 100 105 110
Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser
115 120 125
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser
130 135 140
Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
wherein: Xaa at position 70 is Gin or Glu;
Xaa at position 73 is Asn, Asp or Gin;
Xaa at position 77 is Asn, Asp or Gin;
Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu; Xaa at position 129 is Gin or Glu;
Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
14. A protein of the formula:
80 85 90 Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu
95 100 105
Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val 110 115 120
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
125 130 135
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser
140 Pro Gly Cys
wherein:
Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu; Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
15. A protein of the formula:
90 95 100
Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser
105 110 115
Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val
120 125 130 Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa
135 140
Leu Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 95 is Trp or Gin;
Xaa at position 125 is Gin or Glu;
Xaa at position 129 is Gin or Glu; Xaa at position 131 is lie, Leu, Met or methionine sulfoxide;
Xaa at position 133 is Trp or Gin; and
Xaa at position 134 is Gin or Glu; or a pharmaceutically acceptable salt thereof.
16 A protein of any one of Claims 1 through 15, wherein: Xaa at position 2 is Gin; Xaa at position 17 is Asn; Xaa at position 22 is Thr, Xaa at position 23 is Gin; Xaa at position 29 is Gin; Xaa at position 49 is Met Xaa at position 51 is Gin; Xaa at position 57 is Gin; Xaa at position 58 is Gin; Xaa at position 63 is Met Xaa at position 67 is Asn; Xaa at position 70 is Gin; Xaa at position 73 is Asn; Xaa at position 77 is Asn; Xaa at position 95 is Trp; Xaa at position 125 is Gin; Xaa at position 129 is Gin; Xaa at position 131 is Met; Xaa at position 133 is Trp; and Xaa at position 134 is Gin.
17. A method of treating obesity, which comprises administering to a mammal in need thereof a protein of any one of Claims 1 through 15.
18. A pharmaceutical formulation, which comprises a protein of any one of Claims 1 through 15 together with one or more pharmaceutically acceptable diluents, carriers or excipients therefor.
PCT/US1996/000947 1995-01-31 1996-01-29 Anti-obesity proteins WO1996023514A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP8523609A JPH11501297A (en) 1995-01-31 1996-01-29 Anti-obesity protein
AU47660/96A AU4766096A (en) 1995-01-31 1996-01-29 Anti-obesity proteins
EP96903648A EP0836620A1 (en) 1995-01-31 1996-01-29 Anti-obesity proteins

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US38126695A 1995-01-31 1995-01-31
US38124795A 1995-01-31 1995-01-31
US38104795A 1995-01-31 1995-01-31
US38145195A 1995-01-31 1995-01-31
US08/381,057 US5580954A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,037 1995-01-31
US08/381,049 US5574133A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,370 US5525705A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,163 US5563245A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,666 1995-01-31
US08/381,037 US5563243A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,054 US5569743A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,050 1995-01-31
US08/381,041 1995-01-31
US08/381,040 US5552522A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,041 US5567678A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,163 1995-01-31
US08/381,666 US5521283A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,451 1995-01-31
US08/381,054 1995-01-31
US08/381,050 US5563244A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,057 1995-01-31
US08/381,034 US5532336A (en) 1995-01-31 1995-01-31 Anti-obesity proteins
US08/381,266 1995-01-31
US08/381,040 1995-01-31
US08/381,247 1995-01-31
US08/381,370 1995-01-31
US08/381,034 1995-01-31
US08/381,049 1995-01-31
US08/381,047 1995-01-31
US08/384,492 1995-02-06
US08/383,650 US5691309A (en) 1995-01-31 1995-02-06 Anti-obesity proteins
US08/383,632 US5569744A (en) 1995-01-31 1995-02-06 Anti-obesity proteins
US08/383,649 1995-02-06
US08/383,632 1995-02-06
US08/384,492 US5594104A (en) 1995-01-31 1995-02-06 Anti-obesity proteins
US08/383,649 US5567803A (en) 1995-01-31 1995-02-06 Anti-obesity proteins
US08/383,650 1995-02-06

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AU (1) AU4766096A (en)
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WO1997016550A1 (en) * 1995-11-02 1997-05-09 Bristol-Myers Squibb Company Polypeptide fragments derived from the obese gene product
WO1997046585A2 (en) * 1996-06-06 1997-12-11 Smithkline Beecham P.L.C. Fragments of leptin (ob protein)
WO1998016545A1 (en) * 1996-10-11 1998-04-23 Eli Lilly And Company Therapeutic proteins
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 (en) 1998-10-14 2000-04-20 Amgen Inc. Site-directed dual pegylation of proteins
AU730501B2 (en) * 1996-04-23 2001-03-08 Novozymes A/S Animal feed additives
US6429290B1 (en) 1994-08-17 2002-08-06 The Rockefeller University OB polypeptides, modified forms and derivatives
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