HRP970654A2 - Muteins of obese protein - Google Patents

Description

Obese protein (leptin), a hormone secreted from fat, plays a central role in controlling body fat. Genetic defects, leading to obes protein deficiency (ob/ob mice) or resistance to obes protein (ob), (db/db mice, fa/fa rats), in both cases cause severe obesity (Zhang et al., Nature 372 , 425-431 [1994]; Lee et al., Nature 379, 623-635 [1996]; Chen et al., Cell 84, 546-549 [1996]. In ob/ob mice and WT mice (Campfield et al ., Science 269, 546-549 [1995]; Haalas et al., Science 269, 543-546 [1995]; Pelleymounter et al., Science 269, 540-543 [1995] administration of recombinant ob protein reduces food intake and increases energy expenditure.The effects of reducing body weight are similarly mediated by the signaling leptin receptor in the hypothalamus (Tartaglia et al., Cell 83, 1263-1271 [1995).

Ob mutants with in vivo antagonistic properties have now been discovered. Administration of such mutants results in a progressive reduction in body weight. Ob mutants can therefore be used therapeutically for disorders accompanying chronic disease and weight loss disorders, such as anorexia and cachexia, where weight gain is desirable.

The proposed invention therefore provides mutants with normal receptor binding activity in the competition assay and with an inability to translate biological signals. Preferred ob mutants of the proposed invention are those that include SEQ ID No: 1, where Asp is located at position 127 Ser or Arg is Gln at position 128, with the designations S127D and R128Q, respectively.

The mutants of the proposed invention at position 127 or 128 may contain amino acid residues other than Asp or Gln, respectively, if such substitution does not generally change their binding activity and inability to translate biological signals. The amino acid residues at position 127 and 128 can be selected from the group of α-amino acids generally found in nature, except for the amino acid residues Ser and Arg.

Ob mutants of the proposed invention may contain specific sequences, which preferentially bind to the affinity support material. Examples of such sequences are sequences containing at least two contiguous histidine residues (in this regard, see European Patent No. 282,042). Such sequences bind selectively to nitrile-triacetic acid-nickel chelate resins (Hochuli and Döbeli, Biol. Chem. Hoppe-Seyler 368, 748 [1987]; European Patent No. 253,303). Ob mutants, which contain such a specific sequence, can therefore be selectively isolated from the remaining polypeptides. The specific sequence can be linked to the C-terminus or to the N-terminus of the amino acid sequence of the ob mutant.

Ob mutants may also have one or more chemical units attached to their amino acid sequences, including water-soluble polymers, such as polyethylene glycol. Derivatives derivatized with polyethylene glycol may be mono-, di-, tri- or tetrapegylated, eg N-terminally monopegylated. Preferred pegylated derivatives of ob mutants of the present invention include ob mutants comprising SEQ ID No:1, where Asp is present at position 127 Ser or Gln is present at position 128.

The proposed invention also provides DNA sequences coding for ob mutants of the proposed invention, expression vectors containing these DNA sequences, host cells containing such vectors for producing ob mutants and methods for producing such DNA sequences, recombinant vectors and host cells. Methods for expression, isolation and purification of ob mutants are also described.

Since the entire DNA sequence of the gene encoding the natural ob protein (hereinafter also referred to as human leptin, lH) is known (Zhang et al., supra), the amino acid sequences encoding the ob mutants of the present invention can be chemically synthesized using standard methods known in the art, primarily the solid phase method, such as the methods of Merrifield (J. Am. Chem. Soc. 55, 2149-2154 [1963]). Alternatively, the ob mutants of the proposed invention can be produced using DNA recombinant technology methods (Maniatis et al. in "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory [1982]). First, the DNA sequence corresponding to the ob mutant is cloned from the DNA encoding the native ob protein and then inserted into a suitable expression vector that produces the necessary expression signals.

Expression vectors suitable for use in prokaryotic host cells are listed, for example, in the aforementioned Maniatis et al. Particularly 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]).

Such prokaryotic expression vectors, containing DNA sequences encoding the ob mutants of the present invention, operably linked to a sequence controlling expression, can be incorporated into any suitable prokaryotic host cell using conventional methods. The choice of a suitable prokaryotic host cell is determined by various factors, which are well known in the art. Thus, for example, compatibility with the chosen vector, toxicity of the expression product, expression features, the necessity of biological safety measures and cost all play a role, and a compromise must be found between all these factors.

Suitable prokaryotic host organisms include Gram-negative and Gram-positive bacteria, for example E. coli and B. subtilis species. E.coli subspecies that can be used are E.coli M15 (described as subspecies OZ 291 by Villarejo et al. in J. Bacteriol. 120. 466-474 [1974]) and E.coli W3110 [ATTC No. 27325]. In addition, however, in addition to the above-mentioned E.coli subspecies, other commonly available E.coli subspecies such as E.coli 294 (ATCC No. 31446) and E.coli RR1 (ATCC No. 31343) can also be used. .

Expression vectors suitable for use in mammalian host cells include, but are not limited to, pBC12MI, pBC12BI, pSV2dhFr, p91023(B), pcDV1, pRSVcat, pGA291, pGA293, pGA296, pBC12/HIV/IL-2, and pGA300.

Such vectors are preferably introduced by transfection into suitable mammalian host cells.

Mammalian host cells that can be used include, e.g., human Hela, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, African green monkey CV1 cells, quail QC1-3 cells, Chinese hamster ovary (CHO) cells, mouse L cells and COS cell lines.

The way in which the mutants of the proposed invention are expressed depends on the selected expression vector system and the host cell.

Typically, the prokaryotic host organism, which contains the desired expression vector, is grown under conditions that are optimal for the growth of the prokaryotic host organism. At the end of exponential growth, when the increase in the number of cells per unit of time decreases, the expression of the desired ob mutant is induced, i.e. the DNA that codes for the desired ob mutant is transcribed and the transcribed mRNA is translated. Induction can be carried out by adding an inducer or depressor to the growth medium or by changing a physical parameter, eg by changing the temperature.

Mammalian host cells containing the desired expression vector are grown under conditions that are optimal for growth of the mammalian host cells. A typical expression vector contains a promoter element, which mediates mRNA transcription, a protein coding sequence, and signals necessary for efficient termination and polyadenylation of transcription. Additional elements may include amplification agents and intervening sequences linked by donor and acceptor linkers.

Most vectors, which are used for transient expression of a given coding sequence, carry an SV40 replication source, which allows them to replicate to high numbers in cells (eg COS cells), which significantly amplify the T antigen required to initiate viral DNA synthesis. Transient expression is not restricted to COS cells. Any transfectable mammalian cell line can be used for this purpose. Elements controlling the size of transcription include early or late promoters from SV40 and long terminal repeats (LTRs) from retroviruses, eg RSV, HIV, HTLVI. However, cellular signals (eg the human β-actin promoter) can also be used.

Alternatively, stable cell lines carrying the desired gene embedded in the chromosome can be selected after co-transfection with a selectable marker, such as gpt, dhfr, neomycin or hygromycin.

The transfected gene can now be amplified to amplify large amounts of the foreign protein. A marker that can be used to develop cell lines carrying more than 1000 copies of the desired gene is dihydrofolate reductase (DHRFR). Mammalian cells grow in increasing amounts of methotrexate. Then, when the methotrexate is withdrawn, the cell lines contain an amplified gene embedded in the chromosome.

A baculovirus-insect cell system can also be used to produce the ob mutants of the present invention (for literature see Luclow and Summers, Bio/Technology 6, 47-55 [1988]. Ob mutants produced in insect cells infected with recombinant baculovirus can undergoes post-translational processing involving N-glycosylation (Smith et al., Proc. Nat. Acad. Sci. USA 82, 8404-8408) and O-glycosylation (Thompsen et al., 12th International Herpesvirus Workshop, University of Philadelphia, Pennsylvania).

To isolate small amounts of ob mutants propagated in prokaryotic host cells for analytical purposes, eg, for polyacrylamide gel electrophoresis, the host cells can be disrupted by treatment with a detergent, eg, sodium dodecyl sulfate (SDS). Larger amounts of these mutants can be obtained by mechanical treatment (Charm et al., Meth. Enzymol. 22, 476-556 [1971]), enzymatic (lysozyme treatment) or chemical treatment (with detergent, urea or guanidinium hydrochloride, etc.), then using known methods, e.g. centrifugation at different gravities, precipitation with ammonium sulfate, dialysis (under normal pressure or under reduced pressure), preparative isoelectric focusing, preparative gel electrophoresis or using different chromatographic methods such as gel filtration, high-performance liquid chromatography (HPLC), ion-exchange chromatography, reverse-phase chromatography and affinity chromatography (eg on Sepharose®Blu Cl-6B or on carrier-linked monoclonal antibodies directed against native ob protein and ob mutants of the present invention).

Ob mutants propagated in mammalian host cells or in the baculovirus-insect cell vector system can be isolated from host cell media using standard protein purification methods.

Ob mutants can be used for the preparation of pharmaceutical compositions and for the treatment of disorders accompanying chromic diseases, eg, long-term anorexia, infectious anorexia, cachexia and weight loss disorders, eg, cancer and AIDS, and their applicability in patients with anorexic neurosis can be tested.

They can be administered in pharmaceutically acceptable oral, injectable or topical formulations and procedures. The dosage and frequency of doses may be equivalent to those currently used in clinical applications of natural ob proteins. The pharmaceutical compositions of the proposed invention contain ob mutants, optionally together with a monoclonal antibody against the ob mutant of the proposed invention and a compatible, pharmaceutically acceptable carrier material. Any common carrier material can be used. The carrier material can be organic or inorganic 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. Additives, such as flavor enhancers, preservatives, stabilizers, emulsifiers, buffers and the like, may be added in accordance with accepted pharmaceutical compounding practice.

Pharmaceutical preparations can be made in any conventional manner including: a) a solid form for oral administration such as tablets, capsules, pills, powder, granules and the like; b) 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 surface administration such as solutions, suspensions, ointments, creams, gels, micronized powders, aerosols and the like. Pharmaceutical preparations may be sterilized and/or may contain additional substances such as preservatives, stabilizers, wetting agents, emulsifiers, salts for changing osmotic pressure and/or buffers.

Parenteral dosage forms can be infusion or injection solutions that can be injected intravenously or intramuscularly. These preparations may also contain other medically active substances. Additives, such as preservatives, stabilizers, emulsifiers, buffers and the like, may be added in accordance with accepted pharmaceutical compounding practice.

Furthermore, antibodies can be raised against ob mutants of the proposed invention. These antibodies can be used in a known manner for diagnostic or therapeutic purposes, for purification purposes, and for enhancing the biological effects of ob mutants. Such antibodies may be produced by injecting a mammal or bird with an amount of a vaccine formulation containing an ob mutant of the present invention and a compatible pharmaceutical carrier sufficient to induce antibody formation against said mutant. The appropriate required amount of ob mutant is known to the person skilled in the art or can be determined by routine experimentation. As used in connection with the present invention, the term "pharmaceutical carrier" may mean standard compositions suitable for human administration or typical adjuvants used in animal vaccines.

Suitable adjuvants for animal vaccination include, but are not limited to, Freund's complete or incomplete supplement (not suitable for shell use or for cattle), supplement 65, containing peanut oil, mannide monooleate, and aluminum phosphate and alum; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N1-N-N-dioctadecyl-N'-N-bis(2-hydroxyethylpropanediamine), methoxy-hexadecylglycerol, and pluronic polyols; polyanions such as pyran, dextran sulfate, poly IC, polyacrylic acid, carbopol; peptides such as muramyl dipeptide, dimethylglycine, tuftsin; oil emulsions; and TiterMax. Soluble ob mutants can also be administered after incorporation into liposomes or other microorganisms, or after conjugation to polysaccharides, other proteins or other polymers or in combination with Quil-A, to form “Iscoms” (immunostimulatory complexes) (Morein et al., Nature 308, 457 [1984]). TiterMax supplement (Vaxcell Inc., 3000 Northwoods Parkway, Norcross, GA 30071, USA) is preferred.

Typically, the initial vaccination is given several weeks later with one or more “booster” vaccines, the net effect of which is to produce high titers of antibodies against ob mutants, which can then be harvested in the usual way.

The second method consists in applying the well-known Koehler and Milstein procedure for the production of nomoclonal antibodies. To find different monoclonal antibodies, which are directed against the same antigen but against different epitopes, the method according to Stähli et al. can be applied. (J. of Immunological Methods 32, 297-304 [1980].

Finally, the mutants of the present invention can be used to test methods of identifying ob protein agonists.

It has also been found that the use of leptin or ob mutants of the proposed invention in combination with anti-leptin antibodies, preferably in combination with anti-leptin monoclonal antibodies, enhances the effects of these proteins. Therefore, the proposed invention additionally provides for the use of ob mutants of the proposed invention together with anti-leptin antibodies, especially monoclonal anti-leptin antibodies, for the preparation of pharmaceutical compositions and for the treatment of disorders accompanying chronic diseases, e.g. long-term anorexia and anorexia infectious cachexia, and wasting disorders, e.g. .cancer and AIDS.

Pharmaceutical compositions containing ob mutants of the present invention, together with anti-leptin antibodies, can be formulated and used as described above.

Additionally, the proposed invention provides for the use of human leptin (human obese protein) together with antibodies against leptin, for the preparation of pharmaceutical compositions and for the treatment, prevention and control of obesity and related diseases.

Pharmaceutical compositions containing human leptin together with anti-leptin antibodies can be formulated and used as described in European Patent Application Publication No. 741 187, or as previously described herein.

The preparation of human leptin is known, for example, from European patent application, publication no. 741 187 and UK patent application 2 292 382. The term "human leptin (human obese protein)" includes all types of human leptin described in these patent applications.

Antibodies against human leptin can be produced as described above or as described in European Patent Application No. 741 187 and UK patent application 2 292 382.

This invention, thus far generally described, will be better understood by specific examples, which are included herein for the purpose of illustration without the intention of limiting the invention, unless otherwise indicated, and in connection with the following figures:

Figure 1 shows 125J hL translocation by cold wild-type hL and ob mutants on COS-1 cells transiently transfected with pSVSport-mLRsh. Each point represents ±s.e.m. cpm of bound 125J hL (n = 3).

Figure 2 shows the effects of wild-type hL and ob mutants in the proliferation assay of transfected BA/F3. Data are presented as mean ± s.e.m. cpm of incorporated 3H thymidine.

Figure 3 shows the biological effects of S127D and R128Q in vivo in combination with 2A5 mAb in ob/ob mice. Data are presented as mean change in weight ±s.e.m. (n = 3).

Figure 4 shows the in vivo antagonistic effect of R128Q in combination with 2A5 mAb in C57BL/6 mice. Data are presented as the average change in body weight per group ±s.e.m. (n = 3).

Figure 5 shows the in vivo antagonistic effect of R128Q in combination with 2A5 mAb in C57BL/6 mice. Data are presented as the average change in body weight per group ±s.e.m. (n = 3).

Figure 6 shows the effect of R128Q on serum insulin levels in C57BL/6 mice.

Figure 7 shows the in vivo enhancement effect of hL in ob/ob mice (1 injection/day; 10 µg hL/injection; 1 mg 2A5 mAb/injection). Data are presented as the average change in body weight per group ±s.e.m. (n = 3).

Figure 8 shows the effect of 2A5 mAb on hL in C37/B16 mice. C57/B16 mice were injected intraperitoneally (2 mice/group) with 80 ng hL/10 µg cold 125J-labeled hL with or without 0.2 mg 2A5 mAb.

At the end of the indicated time, the mice were sacrificed and whole blood was collected. 7 µl of serum was analyzed by 15% PAGE and autoradiographed. The signals of one autoradiograph were quantified using a phospho-imager.

Example 1

Identification of mutants

To identify the individual residues involved in receptor binding, 37 single mutations (of which 29 resulted from the change of charged residues to non-charged residues) were created in the human ob protein cDNA using a customized protocol and using commercially available equipment (Transformer, Clontech) and introduced into plasmid pSVSport (BRL). After transfection in COS-1 cells, expression of the mutants in the cell supernatant was quantified by ELISA, using a combination of mAbs and polyclonal rabbit antiserum.

All mutants were tested in a competition experiment by measuring the displacement of 125J hL (prepared by the Iodogen method, Pierce Chemical Co.) from a membrane holding the mouse leptin receptor short form (mLRsh) (Targaglia et al., Cell 83, 1263-1271 [1995]), amplified on the surface of COS-1 cells. 2x106 cells/ml transfected COS-1 cells were incubated for 3-5 hours at 4oC with 1 nM 125J hL, together with various concentrations (2.5 ng/ml - 5 µg/ml) of unlabeled wild-type hL or ob mutants. The bound ligand was separated from the non-radioactive one by centrifugation through a bed of phthalate oil and the γ emission of the precipitate was calculated. All solutions were prepared in Dulbecco's modified Eagle's medium (GibcoBrl) supplemented with 10% FCS, glutamine and gentamicin.

The biological activity was evaluated by an in vivo experiment based on the proliferation of leptin-dependent BA/F3 cells. BA/F3 cells were transfected with a construct encoding a chimeric membrane, which holds a receptor constructed by fusing the extracellular and transmembrane domains of the mouse leptin receptor with the intracellular part of the human βc chain (c stands for common, e. common), a subunit that is essential for signal transduction GM-CFS/IL-3/IL-5 (Tavernier et al., Cell 66, 1175-1184 [1991]). After selection, clones whose growth depends on leptin were obtained. 21F3, a monoclonal that was developed against the related βc chain (referred to as AIC2B; this chain is constitutively present in BA/F3 cells), can be added to reduce background proliferation. After overnight incubation at 37oC without growth factors (= leptin), leptin-dependent BA/F3 cells (200 µl cells; 1x103 cells/spot) were incubated in microtiter 96-well plates with different concentrations of wild-type hL or ob mutants (0 ,05-100 ng/ml). After 72 hours, 0.5 µCi 3H thymidine was added for 4 hours. Cells were harvested and labeled and counted.

Most ob mutants show normal receptor binding and have similar biological activity to the wild-type protein. However, four mutants, R20Q, D40N, S127D and R128Q have different features. Since further detailed investigation depends on the availability of a much purer product, the four mutated genes as well as the wild-type hL were introduced into the pVL1393 vector and cotransfected with linearized baculovirus DNA (Baculogold, PharmMingen) into the genome of Autograpfa californica nuclear polyhedrosis virus in Sf9 cells, as described by Summers and Smith, G.E.A. [1987], Manual of Methods for Baculovirus Vectors and Insect Cell Culture Producers (Texas A & M University). Proteins were purified from insect cell media on a 2A5-mAb column. The identity was assessed on a Coomasie-stained SDS-PAA gel (Laemmli, Nature 227, 680-685 [1970]) while protein concentrations were measured according to Bradford, Anal. Biochem, 72, 248-254 [1976], using BioRad equipment with BSA as standard. Leptin proteins and ob mutants were tested in the translocation experiment (Figure 1), as well as in the BA/F3 proliferation experiment (Figure 2).

The assay enabled the identification of one residue involved in receptor binding, as replacement of arginine at position 20 with glutamine (R20Q) abolished any displacement of 125J hL in the competition experiment (Figure 1). Other purified mutants D40N, S127D, and R128Q were consistently able to translocate the radioligand and in this respect did not differ significantly from wild-type hL, indicating normal receptor binding. However, when the mutants had to be tested in a modified BA/F3 experiment, compared to wild type hL, a reduced biological activity was found (Fig. 2). As expected, R20Q, a mutant lacking receptor interaction, showed no biological effect; the proliferative activity of D40N was reduced by approx. 40%. Surprisingly, almost no proliferative response from S127D was detected in transfected BA/F3 cells, while R128Q showed no biological activity even at a concentration of 100 ngml.

Antagonistic properties of ob mutants S127D and R128Q were further demonstrated in protein-dependent BA/F3 cells, as the proliferative response was completely suppressed in the presence of 10 ng/ml wild-type hL.

Example 2

Biological effects of R127D and R128Q in vivo in ob/ob mice

To evaluate the in vivo effects of the mutants, S127D, constitutively R128Q, wild-type hL and PBS were injected intraperitoneally (i.p) into ob/ob mice together with mAb 2A5. 6-8 week old ob/ob mice were injected with 15 µg wild-type hL, i.e. 15 µg S127D, 15 µg R128Q and PBS (control). Injections were given daily i.p. for 9 days, additionally with mAb 2A5 (1.8 mg per injection). Body weight was measured before the first dose and at the same time every following day. Treatment with wild-type hL protein resulted in significant weight loss (up to 1 g/day), whereas no significant effect was observed in animals injected with S127D, R128Q, and PBS (Fig. 3). On the contrary, a slight increase in body weight could be observed in these cases. An acceptable explanation for this phenomenon is the growth of the eight-week-old mice during the experiment. Taken together, the injections given to ob/ob mice clearly demonstrate the biological inactivity of R128Q and S127D, a fact that is fully consistent with the failure to elicit in vitro proliferative responses in modified Ba/F3 cells (Figure 2).

Example 3

Biological effects of R128Q in vivo in wild-type mice

Nine 9-10-week-old C57/Bl6 mice were divided into three equal groups and injected intraperitoneally with wild-type hL (100 µg per injection) and PBS (control) twice a day (9.300 AM and 5.30 PM) in the presence of 1 .38 mg of 2A5 antibody per injection. Body weight was determined every day by weighing before the first injection at 9:30 AM.

After 15 days, the body weight of the animals was approx. 20% higher than their initial body weight (Figure 4), and after treatment it was stopped and returned to their initial weight (Figure 5). Immediately after the 15-day treatment, the animals from the first experiment (Figure 4) were sacrificed and dissected. Abdominal fat deposits were clearly increased. Blood samples were taken and insulin concentrations were determined by Morgen's method (Diabetes 12, 115 [1963]), using insulin RIA equipment (Linco Research, St. Charles). It can be seen in Figure 6 that, compared to control, insulin levels were significantly elevated in wild-type mice injected with ob mutant R128Q, while injections of hL had the opposite effect. These effects were observed with i.p. injected with wild-type hL in contrast to the data obtained by Schwartz et al., Clin. Invest. 98, 1101-1106 (1996) with rats, where leptin given intracerebroventricularly did not clearly alter insulin levels.

From the above observations, it can be concluded that ob mutants S127D and R128Q behave as leptin antagonists with predominant strong negative effects in vivo.

Example 4

Enhancement of the effect of monoclonal antibodies

Treatment of C57BL/6 mice, which are homozygous (ob/ob) for the obes gene mutation (Zhang et al., supra), with exogenous leptin reduces food intake, increases physical activity, and produces a weight-reducing effect (Halaas et al., supra; Pelleymounter et al., supra; Campfield et al., supra).

Co-injection of recombinant hL with mAb 2A5 has a clear enhancing biological effect in ob/ob mice, meaning that less exogenous leptin is required to achieve the same beneficial effect (weight loss) when co-administered with the antibody (Figure 7).

The observed phenomenon occurs due to the longer lifetime of leptin in the serum, probably due to the stabilization or protective effect caused by antibodies (Figure 8). Monoclonal antibody (mAb) 2A5 is a mouse IgG mAb, which was raised against hL by the known procedure of Koehler and Milstein.

The weight-reducing effect in wild-type mice requires a relatively high dose of exogenous leptin (Halaas et al., Science 269, 543-546 [1995]), clearly not due to the absence of post-translational modifications of the recombinant protein, but as a consequence of pharmacokinetics (Cohen et al. , Nature 382, 589 [1996]). Again, in our experiments the biological effect of exogenously administered hL to C57BL/6 mice (no ob gene mutation) was clearly enhanced by co-injection with mAb 2A5. As can be seen in Figure 4, the animals lost approx. 15% of their initial body weight after 15 days of treatment and maintained this level for the duration of injections with wild-type hL. In another experiment, the animals were treated for nine days and their body weight was recorded further, even after the injections had stopped. From Figure 5 it can be concluded that the animals returned to their original weight. These observations are consistent with the view that leptin acts as a circulatory “real fat messenger” and with the “posited” hypothesis of long-term regulation of energy balance and weight maintenance (Keesey, In Obesity, A. Stunkard, eds. (Philadelphia: W.B. Saunders Co. ), 144-166 [1980]; Harris, R. B. S. FASEB J. 4, 3310-3318 [1990].

Attachment: Printing sequences.

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Claims (21)

1. Ob mutant, characterized by the fact that in the competition test it shows normal receptor binding activity and the inability to translate biological signals. 2. Ob mutant according to claim 1, characterized by the fact that it contains SEQ ID No: 1, where Asp is located at position 127 Ser or Gln is located at position 128 Arg. 3. Ob mutant according to claim 1, characterized in that it is the R128Q protein. 4. DNA sequence, characterized in that it codes for the ob mutant according to claims 1 to 3. 5. An expression vector, characterized in that it includes the DNA sequence according to claim 4 and which is capable of direct expression of the DNA sequence in a compatible prokaryotic or mammalian host cell. 6. A prokaryotic or mammalian host cell, characterized in that it has been transformed with an expression vector according to claim 5. 7. Recombinant baculovirus, characterized in that it contains the DNA sequence according to claim 5. 8. An insect host cell, characterized in that it is infected with a recombinant baculovirus according to claim 7. 9. A method for producing an ob mutant according to claims 1 to 3, characterized in that it includes growing a transformed prokaryotic or mammalian host cell in a suitable medium, so that said mutant is propagated and cleaning said medium. 10. A method for producing an ob mutant according to claims 1 to 3, characterized in that it includes growing an infected host cell from an insect in a suitable medium, so that said mutant is multiplied and cleaning said mutant from the medium of the insect cell. 11. Ob mutant according to claims 1-3, characterized in that it is a therapeutically active agent. 12. Ob mutant according to claims 1-3, characterized in that it is a therapeutically active agent for the treatment of disorders accompanying chronic diseases and weight loss disorders. 13. Pharmaceutical composition, characterized in that it contains the ob mutant according to claims 1-3, if necessary a monoclonal antibody against the obes protein and a compatible pharmaceutically acceptable carrier material. 14. Use of the ob mutant according to claims 1-3, if necessary in combination with a monoclonal antibody against the obes protein, characterized in that it is used for the preparation of pharmaceutical compositions. 15. Use of the ob mutant according to claims 1-3, if necessary in combination with a monoclonal antibody against the obese protein, characterized in that it is used for the preparation of pharmaceutical compositions for the treatment of disorders accompanying chronic diseases and weight loss disorders. 16. Poly- and/or monoclonal antibody, characterized in that it is raised against the ob mutant according to claims 1-3. 17. Ob mutant according to claims 1-3, characterized in that it is produced by the process according to claims 9 and 10. 18. A pharmaceutical composition, characterized in that it contains a human hanging protein together with a monoclonal antibody against said protein and a compatible pharmaceutically acceptable carrier material. 19. The use of a human hanging protein together with a monoclonal antibody against said protein, characterized in that it is used for the preparation of pharmaceutical compositions. 20. The use of human obese protein together with a monoclonal antibody against said protein, indicated by the fact that it is used for the preparation of pharmaceutical compositions for the treatment, prevention or suppression of obesity and associated diseases. 21. Products, pharmaceutical compositions, processes and methods, characterized in that they are substantially in accordance with those described herein.