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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

• Obese protein a fat secreted hormone, plays a central role in the control of body adiposity. Genetic defects that lead to obese protein deficiency (ob/ob mice) or obese protein (ob) resistance (db/db mice, fa/fa rats) both cause severe obesity (Zhang et al., Nature 372, 425-431 [1994]; Lee et al., Nature 379,
• Ob muteins with in vivo antagonistic properties have now been discovered. Administration of such muteins results in progressive increase in body weight.
• the ob muteins are thus of therapeutic use for disorders accompanying chronic diseases and wasting disorders, such as anorexia and cachexia, where weight gain would be beneficial.
• the present invention thus provides ob muteins with normal receptor binding activity in a competition assay and with the inability to transduce biological signals.
• Preferred ob muteins of the present invention are those comprising SEQ ID No: 1, wherein at position 127 Ser is Asp or at position 128
• Arg is Gin, with the designations S127D and R128Q, respectively.
• the ob muteins of the present invention may contain at position 127 or 128 amino acid residues other than Asp or Gin, respectively, if such substitutions do not generally alter their binding activity and inability to transduce biological signals.
• Amino acid residues at position 127 or 128 may be selected from the group of the ⁇ -amino acids commonly found in nature excluding amino acid residues Ser and Arg.
• the ob muteins of the present invention can contain specific sequences that preferably bind to an affinity carrier material. Examples of such sequences are sequences containing at least two adjacent histidine residues
• the ob muteins may also have one or more chemical moieties attached to their amino acid sequences, including water soluble polymers such as polyethylene glycol.
• Polyethylene glycol derivatized derivatives can be mono-, di-, tri- or tetrapegylated, e.g., N-terminal monopegylated.
• Preferred pegylated derivatives of ob muteins of the present invention include ob muteins comprising SEQ ID NO:l, wherein at position 127 Ser is Asp or at position 128 Arg is Gin.
• the present invention also provides DNA sequences which code for the ob muteins of the present invention, expression vectors which contain these DNA sequences, host cells containing such vectors for the production of the ob muteins and processes for the production of such DNA sequences, recombinant vectors and host cells. Methods for the expression, isolation and purification of the ob muteins are also described.
• amino acid sequences coding for the ob muteins of the present invention can be chemically synthesized using standard methods known in the art, preferably solid state methods, such as the methods of Merrifield (J. Am. Chem. Soc. 85, 2149-2154 [1963]).
• the ob muteins of the present invention can be produced using methods of DNA recombinant technology (Maniatis et al. in "Molecular Cloning - A
• a DNA sequence coding for an ob mutein is prepared from DNA coding for the natural ob protein and subsequently incorporated into a suitable expression vector which produces the requisite expression signals.
• prokaryotic host cells suitable for use in prokaryotic host cells are mentioned, for example, in the aforementioned textbook of Maniatis et al. Especially suitable vectors are plasmids of the pDS family (Bujard et al., Methods in Enzymology, eds. Wu and Grossmann, Academic Press, Inc., Vol. 155, 416-433 [1987]).
• prokaryotic expression vectors which contain the DNA sequences coding for the ob muteins of the present invention operatively linked with an expression control sequence, can be incorporated using conventional methods into any suitable prokaryotic host cell.
• the selection of a suitable prokaryotic host cell is determined by different factors which are well-known in the art. Thus, for example, compatibility with the chosen vector, toxicity of the expression product, expression characteristics, necessary biological safety precautions and costs play a role and a compromise between all of these factors must be found.
• Suitable prokaryotic host organisms include gram-negative and gram- positive bacteria, for example E.coli and B.subtilis strains.
• Useful E.coli strains are E.coli M15 (described as strain OZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474 [1974] and E.coli W3110 [ATTC No. 27325]).
• E.coli M15 described as strain OZ 291 by Villarejo et al. in J. Bacteriol. 120, 466-474 [1974] and E.coli W3110 [ATTC No. 27325]
• E.coli 294 ATCC No. 31446
• E.coli RRl ATCC No. 31343
• Expression vectors suitable for use in mammalian hosts cells include but are not limited to pBC12MI, pBC12BI, pSV2dhFr, p91023(B), pcDVl, pRSVcat, pGA291, pGA293, pGA296, pBC12/H ⁇ V/IL-2 and pGA300.
• Such vectors are preferably introduced into suitable mammalian host cells by transfection.
• Mammalian host cells that could be used include, e.g., human Hela, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, CV1 African green monkey kidney cells, quail QC1-3 cells, Chinese hamster ovary (CHO) cells, mouse L cells and the COS cell lines.
• the manner in which the expression of the ob muteins of the present invention is carried out depends on the chosen expression vector/host cell system.
• the prokaryotic host organisms which contain a desired expression vector are grown under conditions which are optimal for the growth of the prokaryotic host organisms.
• the expression of the desired ob mutein is induced, i.e., the DNA coding for the desired ob mutein is transcribed and the transcribed RNA is translated.
• the induction can be carried out by adding an inducer or a derepressor to the growth medium or by altering a physical parameter, e.g., a change in temperature.
• the mammalian host cells which contain a desired expression vector are grown under conditions which are optimal for the growth of the mammalian host cells.
• a typical expression vector contains the promoter element, which mediates the transcription of mRNA, the protein coding sequence, and the signals required for efficient termination and polyadenylation of the transcript. Additional elements may include enhancers and intervening sequences bounded by spliced donor and acceptor sites.
• Most of the vectors used for the transient expression of a given coding sequence carry the SV40 origin of replication, which allows them to replicate to high copy numbers in cells (e.g. COS cells) that constitutively express the T antigen required to initiate viral DNA synthesis.
• Transient expression is not limited to COS cells. Any mammalian cell line that can be transfected can be utilized for this purpose. Elements that control a high efficient transcription include the early or the late promoters from SV40 and the long terminal repeats (LTRs) from retroviruses, e.g. RSV, HIV, HTLVI. However, also cellular signals can be used (e.g. human- ⁇ -actin promoter).
• stable cell lines carrying a gene of interest integrated into the chromosome can be selected upon co-transfection with a selectable marker such as gpt, dhfr, neomycin or hygromycin.
• the transfected gene can be amplified to express large quantities of a foreign protein.
• the dihydrofolate reductase (DHFR) is a useful marker to develop lines of cells carrying more than 1000 copies of the gene of interest.
• the mammalian cells are grown in increasing amounts of methotrexate. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome.
• the baculovirus-insect cell vector system can also be used for the production of the ob muteins of the present invention (for review see Luclow and Summers, Bio/Technology 6, 47-55 [1988]).
• the ob muteins produced in insect cells infected with recombinant baculovirus can undergo post- translational processing including N-glycosylation (Smith et al., Proc. Nat. Acad. Sci. USA 82, 8404-8408) and O-glycosylation (Thomsen et al., 12. International Herpesvirus Workshop, University of Philadelphia, Pennsylvania).
• the host cells can be disrupted by treatment with a detergent, e.g., sodium dodecyl sulphate (SDS).
• a detergent e.g., sodium dodecyl sulphate (SDS).
• SDS sodium dodecyl sulphate
• Larger quantities of these ob muteins can be obtained by mechanical (Charm et al., Meth. Enzymol. 22, 476-556 ]1971]), enzymatic (lysozyme treatment) or chemical (detergent treatment, urea or guanidinium hydrochloride treatment, etc.) treatments followed by use of know methods, e.g.
• the ob muteins expressed in mammalian host cells or in the baculovirus-insect cell vector system can be isolated from the host cell medium using standard protein purification methods.
• the ob muteins can be used for the preparations of pharmaceutical compositions and for the treatment of disorders accompanying chronic diseases, e.g., long-term anorexia, anorexia of infection, cachexia and wasting disorders, e.g., cancer and AIDS and may prove their applicability in patients with anorexia nervosa.
• chronic diseases e.g., long-term anorexia, anorexia of infection, cachexia and wasting disorders, e.g., cancer and AIDS and may prove their applicability in patients with anorexia nervosa.
• the pharmaceutical compositions of the present invention contain ob muteins, optionally in association with a monoclonal antibody against an ob mutein of the present invention and a compatible pharmaceutically acceptable carrier material. Any conventional carrier material can be utilized.
• the carrier material can be an organic or inorganic one suitable for enteral, percutaneous or parenteral administration. Suitable carriers include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene-glycols, petroleum jelly and the like.
• the pharmaceutical preparations may contain other pharmaceutically active agents. Additonal additives such as flavouring agents, preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding.
• the pharmaceutical preparations can be made up in any conventional form including: a) a solid form for oral administration such as tablets, capsules, pills, powders, granules and the like; b) a liquid form for oral administration such as solutions, syrups, suspensions, elixirs and the like; c) preparations for parenteral administration such as sterile solutions, suspensions or emulsions; and d) preparations for topical administrations such as solutions, suspensions, ointments, creams, gels, micronized powders, aerosols and the like.
• the pharmaceutical preparations may be sterilized and/or may contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, salts for varying the osmotic pressure and/or buffers.
• Parenteral dosage forms may be infusions or injectable solutions which can be injected intravenously or intramuscularly. These preparations can also contain other medicinally active substances. Additional additives such as preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding.
• antibodies can be raised against the ob muteins of the present invention. These antibodies can be used in a well-known manner for diagnostic or therapeutic purposes, for purification purposes, and for potentiating biological effects of the ob muteins. Such antibodies can be produced by injecting a mammalian or avian animal with a sufficient amount of a vaccine formulation comprising an ob mutein of the present invention and a compatible pharmaceutical carrier to elicit the production of antibodies against said mutein. The appropriate amount of the ob mutein which would be required would be known to one of skill in the art or could be determined by routine experimentation.
• the term "pharmaceutical carrier” can mean either the standard compositions which are suitable for human administration or the typical adjuvants employed in animal vaccinations. Suitable adjuvants for the vaccination of animals include but are not limited to Freund's complete or incomplete adjuvant (not suitable for human or livestock use).
• Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum phosphate and alum; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, Ni- N-N-dioctadecyl-N'-N-bis(2-hydroxyethylpropanediamine), methoxyhexadecylglycerol, and pluronic polyols; polyanions such as pyran, dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides such as muramyl dipeptide, dimentylglycine, tuftsin; oil emulsions; and TiterMax.
• surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecyl
• the soluble ob muteins could also be administered following incorporation into liposomes or other microcarriers, or after conjugation to polysaccharides, other proteins or other polymers or in combination with Quil-A to form "Iscoms" (immunostimulating complexes) (Morein et al., Nature 308, 457 [1984]).
• the adjuvants TiterMax is preferred (Vaxcel Inc., 3000 Northwoods Parkway, Norcross, GA 30071, USA).
• the initial vaccination is followed some weeks later by one or more "booster" vaccinations, the net effect of which is the production of high titers of antibodies against the ob muteins which can be harvested in the usual way.
• Another method consists in using the well-known Koehler and Milstein technique for producing monoclonal antibodies.
• the method of Stahli et al. J. of Immunological Methods 32, 297-304 [1980] can be used.
• ob muteins of the present invention are useful in screening methods for identifying ob protein agonists.
• the present invention provides in addition the use of the ob muteins of the present invention in association with antibodies against leptin, preferably monoclonal antibodies against leptin, for the preparation of pharmaceutical compositions and for the treatment of disorders accompanying chronic diseases, e.g., long-term anorexia and anorexia of infection cachexia, and wasting disorders, e.g. cancer and AIDS.
• the pharmaceutical compositions containing the ob muteins of the present invention in association with antibodies against leptin may be formulated and utilized as described hereinbefore.
• the present invention provides the use of human leptin (human obese protein) in association with antibodies against leptin, preferably monoclonal antibodies against leptin, for the preparation of pharmaceutical compositions and for the treatment, prevention and control of obesity and associated diseases.
• human leptin human obese protein
• compositions containing the human leptin in association with antibodies against leptin may be formulated and utilized as described in European Patent Application, Publication No. 741 187, or as described hereinbefore.
• human leptin human obese protein
• Antibodies against the human leptin can be produced as described hereinbefore or as described in European Patent Application, Publication No. 741 187 and UK Patent Application 2 292 382.
• Fig. 2 shows effects of wild-type hL and ob muteins in the transfected BA/F3 proliferation assay.
• the data express mean ⁇ s.e.m. cpm incorporated 3 H thymidine.
• Fig. 6 shows the effect of R128Q on the serum insulin levels in C57BL/6 mice.
• Fig. 8 shows the effect of 2A5 mAb on hL in C37/B16 mice.
• C57/B16 mice were injected intraperitonialy (2 mice/group) with 80 ng 125 I labeled hL/10 ⁇ g cold hL with or without 0.2 mg 2A5 mAb. After the indicated time points mice were sacrificed and total blood was taken. 7 ⁇ l of serum was analysed by 15% PAGE and autoradiography. The signals one the autoradiograph were quantified with a phospho-imager.
• mLRsh (Tartaglia et al., Cell 83, 1263-1271 [1995]), expressed on the surface of COS-1 cells.
• 2xl0 6 transfected COS-1 cells/ml were incubated with InM 125 I-hL together with variable concentrations (2.5 ng/ml - 5 ⁇ g/ml) of unlabelled wild-type hL or ob muteins for 3-5 hours at 4°C. Bound ligand was separated from free radioactivity by centrifugation through a phthalate oil cushion and ⁇ emission of the pellet was counted. All solutions were made in Dulbecco's modified Eagle's medium (GibcoBRL) supplemented with 10% FCS, glutamine and gentamycin.
• GibcoBRL Dulbecco's modified Eagle's medium
• BA/F3 cells were transfected with a construct encoding a chimeric membrane anchored receptor which had been constructed by fusing the extracellular and the transmembrane domains of the mouse leptin receptor with the intracellular part of the human ⁇ c chain (c stands for common), a subunit which is indispensable for GM-CSF/IL-3/IL-5 signal transduction (Tavernier et al., Cell 66, 1175-1184 [1991]). After selection clones have been obtained which are leptin dependent for their growth.
• AIC2B a monoclonal which had been developed against the related ⁇ c chain
• AIC2B this chain is constitutively present in BA F3 cells.
• the leptin dependent BA/F3 cells 200 ⁇ l of cells; lxlO 3 cells/point
• variable concentrations of wild-type hL or ob muteins 0.05 - 100 ng/ml.
• 72 h 0.5 ⁇ Ci 3 H thymidine was added for 4 hours. The cells were harvested and the incorporated label counted.
• the antagonistic properties of the ob muteins S127D and R128Q were further illustrated on ob protein dependent BA/F3 cells, since proliferative responses were totally suppressed in the presence of 10 ng/ml wild-type hL.
• S127D, resp. R128Q, wild-type hL and PBS were intraperitoneally (i.p.) injected into ob/ob mice together with mAb 2A5.
• 6-8 week old ob/ob mice were injected with 15 ⁇ g wild-type hL, respectively 15 ⁇ g S127D, 15 ⁇ g R128Q and PBS (control).
• Injections were given daily i.p. for nine days in addition of mAb 2A5 (1.8 mg/injection). Body weight was measured before the first dose and at the same time on each subsequent day.
• mice Nine 9-10 week old C57BL/6 mice were divided in three equal groups and respectively injected intraperitoneally twice-daily (9.30 a.m. and 5.30 p.m.) with wild-type hL (100 ⁇ g/injection), ob mutein R128Q (100 ⁇ g/injection) and PBS (control) in the presence of 1.38 mg antibody 2A5 per injection. Body mass was daily determined by weighing before the first injection at 9.30 a.m.
• NAME F. HOFFMANN-LA ROCHE AG
• B STREET: Grenzacherstrasse 124
• Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr

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• 1997
  • 1997-11-22 WO PCT/EP1997/006541 patent/WO1998024896A2/en not_active Ceased
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