MXPA98007030A - Compositions and methods of ob can protein - Google Patents

Abstract

Canine OB protein and related compositions and methods are provided. Amylacid sequences of canine OB protein, related amino acid sequences, vectors, host cells, methods for recombinant production, related antibodies and / or pharmaceutical compositions, and diagnostic and transgenic animals are provided.

Description

COMPOSITIONS AND METHODS OF OB CANINE PROTEIN FIELD OF THE INVENTION The present invention relates to canine OB protein and related nucleic acid, vectors, host cells, production methods, selective linker molecules, derivatives, pharmaceutical and diagnostic compositions, therapeutic and cosmetic methods.

BACKGROUND Although the molecular basis for obesity is largely unknown, the identification of the "OB gene" and the encoded protein ("OB protein") has yielded something about the mechanisms the body uses to regulate the deposition of body fat. Zhang et al., Nature 372: 425-432 (1994); See also. the Correction at Nature 374: 479 (1995). The OB protein is active in vivo in mutant ob / ob mice (mice obese due to a defect in the production of the OB gene product) in addition to normal, wild-type mice. The biological activity manifests itself. in, among other things, weight loss. See in general f Barinaga, "Obese" Protein Slims Mice, Science 269: 475-476 REF .: 028283 (1995).

It is known, for example, that in mutant ob / ob mice, the administration of OB protein results in a decrease in serum insulin levels and serum glucose levels. It is also known that the administration of OB protein results in a decrease in body fat. This was observed in mutant ob / ob mice, in addition to normal non-obese mice. Pelleymounter et al., Science 269: 540-543 (1995); Halaas et al., Science 269: 543-546 (1995). See also. Campfield et al., Science 269: 546-549 (1995) (Peripheral and central administration of microgram doses of OB protein reduced food intake and body weight of ob / ob and diet-induced obese mice but not in db / db obese mice). In none of these reports have toxicities been observed, even in the highest doses.

The need for treatment for obese animals, particularly pets, is also not established. Obesity could cause cardiovascular problems that affect high levels of blood lipids, arterial plaque, high cholesterol, and high blood pressure. Also, Type II diabetes is associated with obesity. All of these conditions could be present in dogs, particularly dogs fed diets with inappropriate levels of protein or fat.

In addition, it may be desirable or beneficial, particularly for display dogs, to breed dogs that have desired amounts of fat.

Therefore it is desirable to have a cosmetic or therapeutic composition that is safe and effective to administer to dogs for weight loss, fat loss, treatment of hyper- or dyslipidaemia or diabetes.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides the canine OB protein and associated nucleic acids, vectors, host cells, processes for production, related compositions and methods for making and using the same.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1: An alignment of the amino acid sequences for the present canine OB protein (upper sequence, referred to as a pre-protein in reference to the presence of a leader sequence) and the native OB protein of human (Zhang et al. , supra) (the lower one, also referred to as a pre-protein). Using Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA, the similarity in percent is 90,476. Strictly using the amino acid identity, the percent identity is 80,952.

FIGURE 2: An alignment of the amino acid sequences for the present canine OB protein (upper sequence, again referred to as a pre-protein) and native murine OB protein (Zhang et al., Supra) (the lower one, also referred to as a pre-protein). Using the same analysis as in FIGURE 1, above, the similarity in percent is 86,905. Strictly using the amino acid identity is 78,571.

FIGURE 3: An alignment of the amino acid sequences of the OB protein of canine, human, murine and a consensus amino acid sequence is presented. The consensus sequence listed is based on the conserved amino acid regions. The amino acids in the positions that differ between the species are assigned an amino acid on the basis of which amino acid is most prevalent.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides the canine OB protein and associated nucleic acids, vectors, host cells, processes for production, related compositions and methods for making and using the same.

Protein Compositions The full length of the canine OB protein is presented in Sec. ID. No. 1 (later). The leader sequence is (with respect to the numbering of Sec. ID No. 1) -21 to -l. The mature protein consists essentially of (with respect to the numbering of Seq. ID No. 1) 1 to 146. Also, the present OB protein canine could be present or absent the glutamine ("Q") present at position +28 ( with respect to the numeration of the mature protein of Sec. ID No. 1). One could choose to express the mature protein with an N-terminal methionyl residue relative to bacterial expression. Alternatively, one could choose to add the other guide sequences for ease of expression.

Thus, the present canine OB protein could be selected from among the amino acid sequences (with respect to Sec. ID No. 1): (a) -21 to 146 (b) +1 to 146 (c) +1 to 146 with an N-terminal methionine (hereinafter referred to herein as "+1 to 146 set to -1"); Y (d) an amino acid sequence of any of subparts (a), (b) or (c) above, which lacks a glutaminyl residue at position 28.

As will be described more fully below, the present canine OB protein could optionally be formulated with a pharmaceutically acceptable diluent, adjuvant or vehicle.

Compositions and Methods of Nucleic Acid A nucleic acid encoding the canine OB protein is presented in Sec. ID No. 2 (supra). The new nucleic acid sequences include sequences useful in the safe expression in prokaryotic or eukaryotic host cells of the canine OB protein selected from the amino acid sequences (with respect to Sec. ID No. 1): (a) -21 a 146; (b) +1 to 146; (c) +1 to 146 with an N-terminal methionine; Y, (d) an amino acid sequence of any of subparts (a), (b) or (c) above, which lacks a codon encoding a glutaminyl residue at position 28 with respect to Sec. ID No. 1).

The nucleic acids could be purified and isolated, so that the desired coding region is useful for producing the present polypeptides, for example, for diagnostic purposes, as described more fully below. The DNA sequences of the invention specifically comprise: (a) the DNA of Sec. ID No. 2 (supra); (b) the portion of the DNA sequence of Seq. ID No. 2 (supra) which encodes amino acids 1 to 146. DNA sequences encoding variant allelic forms of the canine OB protein, such as the forms encoding the amino acid sequence with the glutamine absent at position 28, and the elaborate DNA sequences encoding the canine OB protein. Such elaborate sequences could easily be constructed according to the "• 8 methods of Alton et al., Published PCT application WO 83/04053.

The genomic DNA encoding the present canine OB protein could contain additional non-coding bases, or 5 introns, and such DNAs; The genomic DNA is obtained by hybridizing all or part of the A'DNCT illustrated in Seq. ID No. 2, to a source of genomic DNA, such as a canine genomic DNA library. Such genomic DNA will encode the functional canine OB protein; however, the use of cDNAs might be more feasible in that, because only the coding region is involved, recombinant manipulation is facilitated.

The present nucleic acid sequences could include the incorporation of "preferred" codons for expression by selected non-mammalian hosts; the supply of cleavage sites by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate the construction of easily expressed vectors.

Also, nucleic acids could be prepared antisense against the DNAs present. Such antisense nucleic acids could be used in modulating the effects of the OB protein in vivo. For example, an antisense nucleic acid could be prepared that impedes the ability of a cell to produce the canine OB protein.

The DNA sequences of the invention are also suitable materials for use as labeled probes in the isolation of human genomic DNA encoding the canine OB protein, as discussed above, and related proteins in addition to the DNA-genomic DNA sequences of other mammal species. DNA sequences could also be used in several alternative methods of protein synthesis (eg, in insect cells) or in gene therapy. The DNA sequences of the invention are expected to be useful in the development of trnsgenic mammal species that could serve as eukaryotic "hosts" for the production of and products in quantity. See, in general, Palmiter et al., Science 222: 809-814 (1983).

Vectors and Host Cells According to another aspect of the present invention, the DNA sequences described herein that encode the canine OB protein are valuable for the information they provide with respect to the amino acid sequence of the mammalian protein which have not been available up to now. Otherwise, the DNA sequences provided by the invention are useful in generating new and useful viral and circular plasmid DNA vectors, new and useful transformed and transfected prokaryotic and eukaryotic host cells (including bacterial and yeast cells and mammalian cells). that grow in culture), and new and useful methods for the growth of culture of such host cells capable of expressing the canine OB protein.

The DNA provided herein (or corresponding RNAs) could also be used for gene therapy for example, treatment of conditions characterized by insufficient expression of the OB protein, such as obesity.

Currently, appropriate vectors for gene therapy (such as retroviral or adenoviral vectors modified for gene therapy purposes and purity and pharmaceutical acceptability) could be administered, for example, for release into the lung. Such vectors could incorporate nucleic acid encoding the present polypeptides for expression at a desired location. Gene therapy could involve a vector that contains more than one gene for a desired protein. Alternatively, a vector could not be used to relatively facilitate stable presence in the host. For example, homologous recombination could facilitate integration into a host genome. (This could be further performed for production purposes, eg, U.S. Patent No. 5,272,071 and WO 91/09955). The nucleic acid could be located within a pharmaceutically acceptable carrier to facilitate cellular entry, such as n 'lipid solution vehicle (e.g., a charged lipid), a liposome vehicle or polypeptide (e.g., polylysine). ). A review article on gene therapy ee Verma, Scientific American, November 1990, pages 68-84 that are incorporated herein by reference.

Methods to produce canine OB protein are also provided herein. Such methods for production include culturing, under appropriate conditions, a host cell containing DNA encoding the canine OB protein. An in vivo population of such host cells is provided herein. Such a host cell could be eukaryotic or prokaryotic, and could contain a vector that includes the DNA encoding the canine OB as provided herein. Alternatively, such a host cell containing DNA from the canine OB protein could have been modified via homologous recombination (see, eg, US Patent No. 5,272,071 and WO 91/09955 as mentioned supra) or other methods for expression altered of the canine OB protein. Then it would be obtained via the collection of the desired canine OB protein. Optionally, such canine OB protein could be purified or further processed and the desired formulations prepared.

Transgenic animals One might wish to produce dogs that have altered canine OB levels. This could be done by preparing transgenic dogs. One way to prepare a transgenic dog is by genetically manipulating a dog embryo with a "transgene", which typically includes a promoter and a desired DNA. In this case, the desired DNA could be selected from those listed above. The promoter could be for tissue-specific expression such as liver-specific expression. Then this transgene would be inserted into the dog's embryo. This could be done by microinjection or other means. One or more such embryos are then implanted in a pseudo-pregnant adoptive dog. The embryo develops and the dog gives birth. An expert in the art will recognize other means of preparing transgenic animals. Such animals could be transgenic to increase the expression of the canine OB protein, thus making animals thinner, or transgenic via the expression of "destroyed" canine OB, for example, using a defective canine OB DNA as a transgene. One or more such animals could be prepared at the same time (eg, multiple embryo implants and multiple births), and such animals could also be reproduced. Thus, transgenic dogs having expression of the altered canine OB protein are provided herein. Such alteration could be in terms of quantity (eg, increased or decreased) or location of the expression of the canine OB protein (eg, tissue-specific expression). The progeny of such transgenic animals are also provided here.

Such animals could be for pets, for exhibition dogs, for breeding purposes, or to provide in vivo bioassays Derivatives, Pharmaceutical Compositions The preend protein could also be derived by the binding of one or more chemical radicals to the protein radical. The chemically modified derivatives could also be formulated for routes of intraarterial, intraperitoneal, intramuscular, subcutaneous, intravenous, oral, nasal, pulmonary, topical or other administration routes. Chemical modification of biologically active proteins have been found to provide additional advantages over certain circumstances, such as increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity. See U.S. Patent No. 4,179,337, Davis et al., Published December 18, 1979. For a review, see Abuchowski et al., In Enzymes as Drugs. (J.S. Holcerberg and J. Roberts, eds., Pp. 367-383 (1981)). A review article describing the modification of protein and fusion proteins is Francis, Focus on Growth Factors 3 .: 4-10 (May 1992) published by Mediscript, Mountview Court, Friern Barnet Lane, London N20, OLD, UK ).

Chemical Radicals for Derivation The chemical radicals suitable for the derivation could be selected from several water-soluble polymers. The selected polymer should be soluble in water so that the protein to which it binds does not precipitate in an aqueous environment, such as a physiological environment. Preferably, for therapeutic use of the preparation of the final product, the polymer will be pharmaceutically acceptable. One skilled in the art will be able to select the desired polymer based on such considerations as if the polymer / protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis and other considerations. For the present proteins, the effectiveness of the derivation could be ascertained by administering the protein or derivative, in the manner intended (eg, by osmotic pumping, or, more preferably, by injection or infusion, or, formulated for oral, pulmonary delivery). or nasal, for example), and observing the biological effects as described to uí.

The water-soluble polymer could be selected from the group consisting of, for example, polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either random or non-random homopolymers or copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyols. polyoxyethylated, polystyrenemaleate and polyvinyl alcohol. Polyethylene glycol propionaldehyde could have processing advantages due to its stability in water. The desired molecular weight of the polymer could be determined empirically, based on the desired characteristics, in addition to the degree of branching of the polymer.

The fusion proteins could be prepared by linking polyamino acids to the OB protein radical. For example, the polyamino acid could be a carrier protein that serves to increase the average circulation life of the protein. For the present therapeutic or cosmetic purpose, such polyamino acid should. be those that do not create neutralizing antigenic response, or other adverse response. Such a polyamino acid could be selected from the group that you did of serum albumin (such as canine serum albumin), an antibody or portion thereof (such as a constant antibody region, sometimes called "Fe") or other polyamino acids. As indicated below, the location of the polyamino acid linkage could be at the N terminus of the OB protein radical, or elsewhere, and could also be connected by a chemical "linker" radical, such as a polyimino acid linker, to the OB protein.

"The number of polymer molecules thus bound could vary, and one skilled in the art will be able to ascertain the effect on the function." It could be mono-derived, or could provide for a di-, tri-, tetra- or some combination by derivation, with the same or different chemical radicals (eg, polymers, such as different weights of polyethylene glycols) The ratio of polymer molecules to protein (or peptide) molecules will vary, depending on their concentrations in the mixture In general, the optimum ratio (in terms of reaction efficiency in which there is no excess of unreacted protein or polymer) will be determined by factors such as the desired degree of derivation (eg, mono-, di-, tri-, etc.), the molecular weight of the selected polymer, whether the polymer is branched or unbranched and the reaction conditions.

The chemical radicals should be bound to the protein with consideration of the effects on the functional or antigenic domains of the protein. There are a number of joining methods available to those skilled in the art. P. ex. EP 0 401 384 incorporated herein by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20: 1028-1035 (1992) (which reports the pegylation of GM-CSF using tresyl chloride). For example, a reactive group, such as a free amino or carboxyl group, could be used. Amino acid residues that have a free amino group could include liein residues and the N-terminal amino acid residue. Those that have a free carboxyl group could include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residues. The sulfhydryl groups could also be used as a reactive group. Binding in important residues to bind the receptor should be avoided if desired in the receptor binding.

One could specifically desire the N-terminal chemically modified protein. Selective N-terminal chemical modification could be performed by reductive alkylation that involves the differential reactivity of the different types of primary amino groups (lysine versus N-terminal) available for derivation in a particular protein. Under the appropriate reaction conditions, the substantially selective derivation of the protein at the N-terminus with a carbonyl group containing the polymer is achieved. For example, the N-terminal protein could be selectively pegylated (polyethylene glycolized) by performing the reaction at a pH that allows taking advantage of the pKa differences between the e-amino group of the lysine residues and that of the a-amino group of the residue N-terminal protein. By such selective derivation, the binding of the water-soluble polymer to a protein is controlled: the conjugation with the polymer is carried out predominantly at the N-terminus of the protein and no significant modification of the other reactive groups, such as groups, is present. amino of side chain of lysine. Using reductive alkylation, the water-soluble polymer could be of the type described above, and should have a simple reactive aldehyde for coupling to the protein.

. * A derivative is preferred (pa polierdl? Licoliza ±.) N-tepran ^ cara -far-dlidad in the n-d-rraón of a therapeutic. The N-terminal p3gj] _ac-Í £ n (poHetdleng1 i mi i V ^ G? F?) Ensures a homogeneous product as the characterization of the product is simplified in relation to di-, tri-, or other multi-pegged products. The use of the above reductive alkylation process for the preparation of an N-terminal product is preferred for ease in commercial processing.

Pharmaceutical Compositions In yet another aspect of the present invention, methods are provided that use pharmaceutical compositions of the proteins and derivatives. Such pharmaceutical compositions could be for administration by injection or oral, pulmonary, nasal, transdermal or other forms of administration. In general, pharmaceutical compositions comprising effective amounts of the protein or products derived from the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or vehicles are comprised by the invention. Such compositions include diluents of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and energizing agents (eg, Tween 80, Polysorbate 80), antioxidants (eg, ascorbic acid, eodium metabisulfite), pre-preservatives (eg, Thimereol, benzyl alcohol) and bulky agents (eg, lactose, mannitol); the incorporation of the material into particulate preparations of polymeric compounds such as polyacetic acid, polyglycolic acid, etc. or in liposomes. Hyaluronic acid could also be used, and this could have the effect of promoting prolonged duration in the circulation. Such compositions could influence the physical state, the stability, the release rate in vivo, and the in vivo clearance rate of the present proteins and derivatives. See, p. ei. , Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are incorporated herein by reference. The compositions could be prepared in liquid form, or in the form of dry powder, such as the lyophilized form. Implantable prolonged-release formulations, such as transdermal formulations, are also contemplated.

Solid oral dosage forms are contemplated herein, which are generally described in Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) in Chapter 89, which is incorporated herein by reference. Solid dosage forms include tablets, capsules, pills, lozenges, granules. Liposomal or proteinoid encapsulation could also be used to formulate the present compositions (such as, for example, proteinoid microeffeins reported in U.S. Patent No. 4,925,673). Liposomal encapsulation could be used and liposomes could be derived with various polymers (e.g., U.S. Patent No. 5,013,556). A description of the possible dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and C.T. Rhodes Chapter 10, 1979, incorporated herein by reference. In general, the formulation will include the protein (or analogue or derivative), and inert ingredients that allow protection against the stomach environment and release of the biologically active material in the intestine.

Oral dosage forms of the proteins derived above are also specifically contemplated. The protein could be chemically modified so that the oral release of the derivative is effective. In general, the contemplated chemical modification is the binding of at least one radical to the protein X molecule (or peptide), wherein said radicals allow (a) inhibition of proteolysis; and (b) entry into the bloodstream of the stomach or intestine. It is also desired to increase the overall stability of the protein and increase in circulation time in the body. Examples of such radicals include: Polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abichoweki and Davis, Soluble Polymer-Enzyme Adducts. In: "Enzymes as Drugs", Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, (1981), pp. 367-383; Newmark, et al., J. Appl. Biochem. 4: 185-189 (1982). Other polymers that could be used are poly-1,3-dioxane and poly-1,3,6-thioxocane.

For the protein (or derivative) the location of the release could be the stomach, the small intestine (the duodenum and jejunum or the ileum) and the large intestine. An expert in the art has available formulations that will not die in the stomach, yet will release the material in the duodenum or in any part of the inteetin. Preferably, the release will avoid the harmful effects of the stomach environment, either by protection of the protein (or derivative) or. by release of biologically active material beyond the stomach environment, such as in the intestine.

To ensure total gastric resistance, a coating impervious to at least pH 5.0 is essential. Examples of the most common ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCPSS polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S and Shellac. These coatings could be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not deaceted for, protection against the stomach. This may include sugar coatings or coatings that make the tablet easier to ingest. The capsules could consist of hard coating (such as gelatin) for release of the dry therapeutic p. ex. dust; For liquid forms, you could use a soft coating of gelatin. The capsule coating material could be coarse starch or other edible paper. For pills, pills, molded tablets or crushed tablets, wet mass techniques can be used.

The therapeutic can be included in the formulation as fine ultiparticulates in the form of granules or pills of approximately 1 mm particle size. The formulation of the material for capsule administration can also be as a powder, slightly compressed fillings or even as tablets. The therapeutic could be prepared by compression.

The coloring and flavoring agents could also be included. For example, the protein (or derivative) could be formulated (such as by liposomal encapsulation or microsphere) and then further contained within an edible product, such as a food or beverage.

The volume of the therapeutic could be diluted or increased with an inert material. These diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, eucalypt, modified dextrans, and starch. Certain inorganic salts could also be used as fillers that include calcium trifoephate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emco prese and Avicel.

The disintegrants could be included in the formulation of the therapeutic in a solid dosage form. Materials used as disintegrants include but are not limited to starch which includes the commercial starch based disintegrant, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramilopectin, sodium alginate, gelatin, orange peel, carboxymethyl cellulose acid, natural sponge and bentonite could be included. Another form of the disintegrants are insoluble cation exchange resins. Powdered gums could be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth.

Alginic acid and sodium salt are also useful as disintegrants.

Binders could be used to maintain the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could be used in alcoholic solutions to granulate the therapeutic.

An antifrictional agent could be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants could be used as a layer between the therapeutic and the mold wall, and this may include but is not limited to; Stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants could also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Slippers could be added which could improve the flow properties of the drug during formulation and to help rearrange during compression. The glidants could include starch, talc, fumed silica and hydrated silicoaluminate.

To aid in the dissolution of the therapeutic in the aqueous environment, a surfactant could be added as a moisturizing agent. The surfactants could include anionic detergents such as sodium lauryl sulfate, dioctyl sulfosuccinate and eodium dioctyl sulfonate. Cationic detergents could be used and could include banzalconium chloride or benzethonium chloride. The list of non-ionic potential detergents that could be included in the formulation as surfactants are lauro acrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated, castor oil 10, 50 and 60 glycerol molasses, polysorbate 40, 60, 65 and 80 , sacaroea of fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.

The additives that potentially enhance the entry of the protein (or derivative) are for example oleic acid, linoleic acid and fatty acid linolenic acid.

The controlled release formulation may be desirable. The drug could be incorporated in an inert matrix that allows release either by diffusion or leachate mechanisms p. ex. gums Matrices that degenerate slowly could also be incorporated into the formulation.

Another form of controlled release of this therapeutic is through a method based on the Oros therapeutic system (Alza Corp.), p. ex. The drug is enclosed in a semipermeable membrane that allows water to enter and press the drug through a single small opening due to the osmotic effects. Some enteric coatings also have a prolonged release effect.

Other coatings could be used for the formulation. These include a variety of sugars that could be applied in a coating container. The therapeutic agent could also be given in a film-coated tablet and the materials used in this embodiment are divided into two groups. The first are non-enteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, providone and the polyethylene glycols. The second group consists of enteric materials that are commonly ethers of phthalic acid.

You could use a mixture of materials to provide the optimal coating film. The coating film could be carried out in a coating vessel or in a fluidized bed or by compression coating.

Lung release of the present protein, or derivative thereof, is also contemplated herein. The protein (derivative) is released into the lungs of the mammal while it is inhaled and travels through the pulmonary epithelium covering the bloodstream. (Other reports of this include Adjei et al., Pharmaceutical Research 7: 565-569 (1990), Adjei et al., International Journal of Pharmaceutics 62: 135-144 (1990) (leuprolide acetate), Braquet et al., Journal of Cardiovascular Pharmacology 13. (suppl 5): s 143-146 (1989) (endotelin-1); Hubbard et al., Annale of Ineternal Medicine 3: 206-212 (1989) (al-antitrypsin); Smith et al., J. Clin. Invest. 84: 1145-1146 (1989) (a-1-proteinase); Oswein et al. , "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, 1990 (recombinant human growth hormone); Debs et al., The Journal of Immunology 140: 3482-3488 (1988) (interferon-g and tumor necrosis factor alpha) and Platz et al., U.S. Pat. No. 5,284,656 (gramulocyte colony stimulating factor). Naeal release of the protein (or derivative) is also contemplated.

Release is also contemplated via transport through the mucous membranes.

An expert in the art will be able to ascertain the effective dosages by administration and observation of the desired therapeutic effect. Preferably, the formulation of the molecule will be such that it is between about 0.10 μg / kg / day and 1 g / kg / day will produce the desired therapeutic effect. Effective dosages could be determined using additional time diagnostic tools. For example, a diagnosis to measure the amount of OB protein in the blood (or plasma or serum) could be used first to determine the endogenous levels of the OB protein. Such a diagnostic tool could be in the form of an antibody test, such as an antibody overlay. The amount of endogenous OB protein is quantified initially, and the baseline is determined. The therapeutic dosages are determined as the quantification of the endogenous and exogenous OB protein (ie, the protein, analog or derivative found within the body, whether produced by itself or administered) is continued during the course of therapy. The dosages therefore could vary during the course of therapy, with a relatively high dosage that is used initially, until the therapeutic benefit is observed, and the lower dosages used to maintain the therapeutic benefits.

Selective Linker Molecules A further embodiment of the invention are selective linker molecules, such as monoclonal antibodies that selectively bind canine OB protein. The hybridoma technique originally described by Kohler and Milstein Eur. J. Immunol. 6, 511-519 (1976) has been widely applied to produce hybrid cell lines that secrete high levels of monoclonal antibodies against many specific antigens. Receptor antibodies could also be prepared, (see Huse et al., Science 246: 1275 (1989)). Such recombinant antibodies could also be modified, such as by modifying the complementary determining regions to increase or alter the affinity, or alteration of the constant regions to resemble canine constant regions of such antibodies. Such antibodies, for example, could be incorporated into a kit for diagnostic purposes. Diagnostic equipment could be employed to determine the location and / or amount of canine OB protein of an individual. The < Diagnostic equipment, such as for a sandwich type test, could also be used to determine if an individual has receptors that bind canine OB protein, or those that, to varying degrees, have reduced binding ability or ability. As stated below, such antibodies could be prepared using immunogenic portions of the canine OB protein, particularly the immunogenic portions that are unique to the canine OB protein.

Methods of Use Therapeutic. Therapeutic uses include weight reduction, the treatment or prevention of diabetes, reduction of blood lipids (and treatment of related conditions), increase muscle mass and increase senescence to insulin. In addition, the present compositions could be used for the manufacture of one or more drugs for the treatment or improvement of the above conditions.

Weight Reduction The present compositions and methods could be used for weight reduction. As demonstrated in the murine models (see above), administration of the present OB protein results in weight loss. The weight loss is mainly from adipose tissue, or fat. Such weight loss can be associated with the treatment of concomitant conditions, such as later ones and therefore constitute a therapeutic application. In addition, cosmetic uses are provided herein if weight loss is solely for the improvement in appearance, such as for display dogs or dogs used for breeding.

Treatment of Diabetes. The present compositions and methods could be used in the prevention and treatment of Type II diabetes. As type II diabetes can be correlated with obesity, the use of the present invention to reduce weight can also alleviate or prevent the development of diabetes. In addition, even in the presence of sufficient dosages to result in weight loss, the present compositions could be used to prevent or ameliorate diabetes.

Reduction of blood lipid. Lae present compositions and methods could be used in the modulation of blood lipid levels. Ideally, in situations where reduction is desired only at blood lipid levels, or where maintenance of reduced lipid levels is desired, the dosage will be insufficient to result in weight loss. Thus, during an initial course of therapy of an obese animal, the dosages could be administered according to which the decrease in weight loss and the concomitant blood lipid level is achieved. Once sufficient weight loss is achieved, a sufficient dosage could be administered to prevent re-gain of weight, still sufficient to maintain the desired blood lipid levels. These dosages can be determined empirically, as the effects of the OB protein are reversible. E.g . , Campfield et al., Science 269: 546-549 (1995) at 547. Thus, if a dosage resulting in weight loss is observed when weight loss is not desired, a lower dosage would be administered to achieve lipid levels of blood, even to maintain the desired weight.

Increased Muscle Mass or Insulin Sensitivity Ideally, in situations where only an increase in body mass is desired, the dosage will be insufficient to result in weight loss. Thus, during an initial course of therapy of an obese person, the dosages could be administered according to which the weight loss and the decrease of fat tissue / increase of muscle mass is achieved. Once sufficient weight loss is achieved, a sufficient dosage could be administered to prevent weight re-gain, still sufficient to maintain the desired increase in muscle mass (or, prevention of muscle mass reduction). These dosages can be determined empirically, as the effects of the OB protein are reversible. . ex. , Campfield et al., Science 269: 546-549 (1995) at 547. Thus, if a dosage resulting in weight loss is observed when weight loss is not desired, a lower dosage would be administered to achieve the desired increase in muscle mass, even to maintain the desired weight. To increase the sensitivity to an individual's insulin, consideration of deficiency and eimilaree could be taken into account. It could achieve efficient increase in muscle tissue without weight loss to decrease the amount of insulin (or, potentially, amylin or other potential diabetes treatment drugs) that would be administered to an individual for the treatment of diabetes. To increase the overall strength, there could be considerations of similar dosages. Increased muscle tissue with concomitant increase in overall strength could be achieved with insufficient dosages to result in loss of muscle. Other benefits, such as an increase in red blood cell (and oxygenation in the blood) and a decrease in bone resorption or osteoporosis could also be achieved in the absence of weight loss.

Combination Therapies The present compositions and methods could be used in conjunction with other therapies, such as diet and exercise altered. Other medications, such as those used to treat diabetes (eg, insulin, and possibly amylin), medications that lower cholesterol and blood pressure (such as those that lower blood lipid levels or other cardiovascular drugs), medications that increase activity (eg, ampheta inas), and appetite suppressants. Such an administration could be simultaneous or it could be in seriatim. In addition, the above methods could be used in conjunction with surgical procedures, such as cosmetic surgeries designed to alter the overall appearance of the body (eg, liposuction or laser surgeries designed to reduce body mass, or implant surgeries designed to increase appearance of body mass). The health benefits of cardiac surgeries, such as short circuit surgeries or other surgeries designed to alleviate a deteriorating condition caused by blockage of blood vessels by fatty deposits, such as arterial plaque, could be increased with concomitant use of the presentee compoeicionee and metode. Methods for removing gallstones, such as ultrasonic or laser methods, could also be used either before, during or after a course of the present therapeutic methods. In addition, the present methods could be used as an auxiliary for surgeries or therapies for broken bones, damaged muscle, or other therapies that would be improved by an increase in muscle mass.

Diagnosis The present canine OB protein and nucleic acids could be used for diagnostic purposes. For example, RNAs and DNAs could be used to characterize or detect defects in a gene product or canine OB protein DNA gene of an individual. For example, an obese individual may have a defect in the canine OB gene. The present DNAs could be used to hybridize with the nucleic acid of an individual to detect such defects, such as via PCR technique. The canine OB protein could be used to characterize an OB protein of an individual for its stability to bind the OB receptor, or for other biological activity. For example, a test for the ability of the OB protein to alter lipid metabolism could be prepared by preparing a population of cells containing lipids expressing the OB receptor, and contacting the OB protein with such a population of cells. It could monitor the modulation of lipid content, lipid characteristics or other characteristics. For diagnostic purposes, the present protein or nucleic acids could be associated with a detectable labeled substance such as a radioactive isotope, a fluorescent or chemiluminescent chemical, or another marker appropriate to one skilled in the art. Such nucleic acids could be used for tissue distribution tests (e.g., to detect the transcriptor distribution of canine mRNA OB in different tissue types) or for other tests to determine the location of the OB receptor.

SEQUENCES OF AMINO CIDO AND DNA The following are lists of sequences for the present canine OB protein and related DNA.

For the amino acid sequence (Seq ID No. 1), the first amino acid of the mature protein is in the +1 position and is a valine ("V") and ee indicates in bold. A methionyl residue could be added in position -1, with the elimination of the base sequence, for ease of expression in bacterial systems. Alternatively, one could have other N-terminal modifications, such as recognition sites for agents that cut amino acids, for ease in the production of the protein without the N-terminal methionyl residue. For example, a recognition site could be included in the N term for ease of production (abbreviated as "1-146 set to -1"). In addition, a version with glutamine could be prepared in the absent position 28.

As previously established, the DNA (Sec. ID No. 2) not yet altered from the amino acid sequence being encoded could be altered. Such alterations could be, for example, to include recognition sites for ease of duplication or insertion of the vector, or to include preferred codons for expression in certain systems, such as bacterial expression or eukaryotic expression.

Canine OB protein (Seq. ID No. 1) -21 MRCGPLCRFL WLWPYLSCVE AVPIRKVQDD TKTLIKTIVA RINDISHTQS VSSKQRVAGL DFIPGLQPVL SLSRMDQTLA IYQQILNSLH SRNWQISND 80 LENLRDLLHL LASSKSCPLP RARGLETFES LGGVLEASLY STEWALSRL 130 QAALQDMLRR LDLSPGC * Canine OB Protein DNA (Seq ID No. 2) 1 ATGCGTTGTG GACCTCTGTG CCGATTCCTG TGGCTTTGGC CCTATCTGTC 5 51. CTGTGTTGAA GCTGTGCCAA TCCGAAAAGT CCAGGATGAC ACCAAAACCC 101 TCATCAAGAC GATTGTCGCC AGGATCAATG ACATTTCACA CACGCAGTCT 151 GTCTCCTCCA AACAGAGGGT CGCTGGTCTG GACTTCATTC CTGGGCTCCA 201 ACCAGTCCTG AGTTTGTCCA GGATGGACCA GACGTTGGCC ATCTACCAAC 251 AGATCCTCAA CAGTCTGCAT TCCAGAAATG TGGTCCAAAT ATCTAATGAC 301 CTGGAGAACC TCCGGGACCT TCTCCACCTG CTGGCCTCCT CCAAGAGCTG 351 CCCCTTGCCC CGGGCCAGGG GCCTGGAGAC CTTTGAGAGC CTGGGCGGCG 401 TCCTGGAAGC CTCACTCTAC TCCACAGAGG TGGTGGCTCT GAGCAGACTG 451 CAGGCGGCCC TCCAGGACAT GCTTCGGCGG CTGGACCTCA GCCCTGGGTG 501 CTGA 25 While the present invention has been described in terms of the preferred modalities, it is understood that variations and modifications will be presented to those skilled in the art. Therefore, it is intended that the appended claims cover all equivalent variations that fall within the scope of the invention as claimed.

LIST OF SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: Hernday, Natasha (ii) TITLE OF THE INVENTION: Compositions and Methods of the Canine OB Protein (iii) SEQUENCE NUMBER: 5 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESS: Amgen Inc. (B) STREET: One Amgen Center Drive (C) CITY: Thousand Oaks (D) STATE: California (E) COUNTRY: US (F) C.P. 91320 (v) AVAILABLE COMPUTER FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATE: (A) APPLICATION NUMBER: US 08 / 609,408 (B) PUBLICATION DATE: 01-March-1996 (C) CLASSIFICATION: (viii) ATTORNEY / MANDATORY INFORMATION: (A) NAME: Eggert, Joan D. (C) REFERENCE NUMBER / REGIS RO: A-387 (2) INFORMATION FOR SEC ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 167 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) CHARACTERISTICS: (A) NAME / KEY: Base Sequence (B) LOCATION: -21 a - 1 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 Met Arg Cys Gly Pro Leu Cys Arg Phe Leu Trp Leu Trp Pro Tyr Leu -20 - 15 - 10 Ser Cys Val Glu Ala Val Pro lie Arg Lys Val Gln Asp Asp Thr Lys • 5 1 5 10 Thr Leu lie Lys Thr lie Val Wing Arg lie Asn Asp lie Ser His Thr 15 20 25 Gln Ser Val Ser Ser Lys Gln Arg Val Wing Gly Leu Asp Phe lie Pro 30 35 40 Gly Leu Gln Pro Val Leu Ser Leu Ser Arg Met Asp Gln Thr Leu Wing 45 50 55 lie Tyr Gln Gln lie Leu Asn Ser Leu His Ser Arg Asn Val Val Gln 60 65 70 75 Xle Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Wing 80 85 90 Be Ser Lys Be Cys Pro Leu Pro Arg Wing Arg Gly Leu Glu Thr Phe 95 100 105 Glu Ser Leu Gly Gly Val Leu Glu Wing Ser Leu Tyr Ser Thr Glu Val 110 115 120 Val Ala Leu Ser Arg Leu Gln Ala Ala Leu Gln Asp Mßt Leu Arg Arg 125 130 135 Leu Asp Leu Ser Pro Gly Cys • 140 '145 (2) INFORMATION FOR SEC ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 504 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: ATGCGTTGTG GACCTCTGTG CCGATTCCTG TGGCTTTGGC CCTATCTGTC CTGTGTTGAA 60 GCTGTGCCAA TCCGAAAAGT CCAGGATGAC ACCAAAACCC TCATCAAGAC GATTGTCGCC 120 AGGATCAATG ACATTTCACA CACGCAGTCT GTCTCCTCCA AACAGAGGGT CGCTGGTCTG 180 GACTTCATTC CTGGGCTCCA ACCAGTCCTG AGTTTGTCCA GGATGGACCA GACGTTGGCC 240 ATCTACCAAC AGATCCTCAA CAGTCTGCAT TCCAGAAATG TGGTCCAAAT ATCTAATGAC 300 CTGGAGAACC TCCGGGACCT TCTCCACCTG CTGGCCTCCT CCAAGAGCTG CCCCTTGCCC 360 CGGGCCAGGG GCCTGGAGAC CTTTGAGAGC CTGGGCGGCG TCCTGGAAGC CTCACTCTAC 420 TCCACAGAGG TGGTGGCTCT GAGCAGACTG CAGGCGGCCC TCCAGGACAT GCTTCGGCGG 480 504 CTGGACCTCA GCCCTGGGTG CTGA (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 167 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) FEATURE: (A) NAME / KEY: Base Sequence (B) LOCATION: -21 to -1 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3 Met His Trp Gly Thr Leu Cys Gly Phß Leu Trp Leu Trp Pro Tyr Leu -20 -15 -10 Phe Tyr val Gln Ala val Pro He Gln Lys Val Gln Asp Asp Thr Lys -5 1 5"10 Thr Leu He Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr 15 20 25 Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe He Pro 30 35 40 Gly Leu His Pro He Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala t Val Tyr Gln Gln He Leu Thr Ser Mßt Pro Ser Arg Asn Val He Gln 60 65 70 75 He Be As Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Wing 85 85 '90 Phß Ser Lys Ser Cys His Leu Pro Trp Wing Ser Gly Leu Glu Thr Leu 95 100 105 Asp Ser Leu Gly Val Leu Glu Wing Ser Gly Tyr Ser Thr Glu Val 110 115 120 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln 125 130 135 Leu Asp Leu Ser Pro Gly Cys 140 145 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 167 amino acids (B) TYPE: amino acid 5. (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) FEATURE: (A) NAME / KEY: Base Sequence 0 (B) LOCATION: -21 to -1 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4 Met C s Trp Arg Pro Leu Cys Arg Phe Leu Trp Leu Trp Ser Tyr Leu -20 -15 -? or Ser Tyr Val Gln Ala Val Pro He Gln Lys Val Gln Asp Asp Thr Lys -5 1 5? Thr Leu He Lys Thr He Val Thr Arg He Asn Asp He Ser His Thr 15 20 25 Gln Ser Val Be Ala Lys Gln Arg Val Thr Gly Leu Asp Phe He Pro 30 35 40 Gly Leu His Pro He Leu Ser Leu Ser Lys Mss Asp Gln Thr Leu Ala 45 50 55 Val Tyr Gln Gln Val Leu Thr Ser Leu Pro Ser Gln Asn Val Leu Gln 50 65 70 75 He Wing Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Wing 80 85 90 Phe Ser Lys Ser Cys Ser Leu Pro Gln Thr Ser Gly Leu Gln Lys Pro 95 100 105 Glu Ser Leu Asp Gly Val Leu Glu Wing Ser Leu Tyr Ser Thr Glu Val 110 115 120 Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp He Leu Gln Gln 125 1 0 135 Leu Asp to Ser Pro Glu Cys NFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 167 amino acids (B) TYPE: amino acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) FEATURE: (A) NAME / KEY: Base Sequence (B) LOCATION: -21 to -1 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Met Xaa Trp Gly Pro Leu Cys Arg Phe Leu Trp Leu Trp Pro Tyr Leu - 20 - 15 - 10 Ser Tyr Val Gln Ala Val Pro He Gln Lys Val Gln Asp Asp Thr Lys - 5 - 1 5 10 Thr Leu He Lys Thr lie Val Thr Arg He Asn Asp He Ser His Thr 15 20 25 Gln Ser Val Ser Ser Lys Gln Arg Val Thr Gly Leu Asp Phe He Pro 30 35, 40 Gly Leu His Pro He Leu Ser Leu Ser Lys Met Asp Gln Thr Leu Wing 45 50 55 Val Tyr Gln Gln He Leu Thr Ser Leu Pro Ser Arg Asn Val Xaa Gln 60 65 70 75 He Be As Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu Wing 80 85 - 90 Phe Ser Lys Ser Cys Xaa Leu Pro Xaa Wing Ser Gly Leu Glu Thr Xaa 95 100 105 Glu Ser Leu Gly Val Leu Glu Wing Ser Leu Tyr Ser Thr Glu Val 110 115 120 Val Ala 'Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Xaa Gln 125 130 135 Leu Asp Leu Ser Pro Gly Cys 140 145 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it refers.

Having described the invention as above, the content of the following is claimed as property.

Claims (14)

1. A canine OB protein, characterized in that it is selected from among the amino acids (according to the numbering of Seq. ID No. 1): (a) -21 to 146 (b) +1 to 146 (c) +1 to 146 set to -1; Y, (d) a canine OB protein of any of subparts (a), (b) or (c) above, which lacks a glutaminyl residue at position 28; optionally in a pharmaceutically acceptable diluent, adjuvant or vehicle.

2. A purified and isolated DNA encoding a canine OB protein, characterized in that it is selected from amino acids (according to the numbering of Seq ID No. 1): (a) -21 to 146 (b) +1 to 146 (c) +1 to 146 set to -1; Y, (d) a canine OB protein of any of subparts (a), (b) or (c) above, which lacks a glutaminyl residue at position 28;

3. A DNA of claim 2, characterized in that it is a cDNA.

4. A DNA of claim 2, characterized in that it is a genomic DNA.

5. A DNA of claim 2, characterized in that it is selected from (according to Sec. ID No. 2): (a) the portion of Sec. ID No. 2 that encodes canine OB protein -22 to +146 (according to Sec. ID No. 1); (b) the portion of Sec. ID No. 2 that encodes canine OB protein +1 to +146 (according to Sec. ID No. i); (c) the DNA of subpart (b) which also encodes a methionyl residue at position -1; Y, (d) the DNA of any of subparts (a), (b) or (c) above that lack a codon for glutamine at position 28 with respect to Sec. ID No. 1.

6. A vector, characterized in that it contains a DNA according to any of claims 2, 3, 4 or 5.

7. A eukaryotic or prokaryotic host cell, characterized in that it contains a DNA according to any of claims 2, 3, 4 or 5; or containing a vector containing a DNA according to any of claims 2, 3, 4 or 5.

8. A process for producing canine OB protein, characterized in that it comprises culturing, under appropriate conditions, a population of prokaryotic or eukaryotic host cells containing a DNA according to any of claims 2, 3, 4 or 5, and obtaining the protein Canine OB produced.

9. A selective linker molecule, characterized in that it comprises selectively binding the canine 0B protein.

10. A monoclonal antibody, characterized in that it selectively binds canine OB protein.

11. An equipment characterized in that it contains a selective linker molecule that selectively binds the canine OB protein.

12. A method of treating a dog, characterized in that it comprises administering a therapeutically effective amount of the canine OB protein according to claim 1.

13. A method of claim 12, characterized in that the dog is treated for obesity, type II diabetes, high levels of blood lipids, or to increase muscle mass.

14. A transgenic dog, characterized in that it has the expression of the altered canine OB protein.