Derivatives of s-protein

 

(57) Abstract:

The invention relates to medicine, namely the derived protein S obesity with long half-life. The invention in particular relates to S-protein-immunoglobulin chimeras and polyethylene glycol (PEG)-S-derivatives, which have a long half-life compared to native S-proteins. In addition, the invention relates to a method of reducing appetite and/or weight and the treatment of other physiological States using S-derivatives with a long half-life. The technical result of the invention is the expansion of the means to combat obesity. 12 C. and 14 C. p. F.-ly, 33 ill.

The invention relates to a derivative of OB protein with a long half-life. In particular, the invention relates to OB-protein-immunoglobulin chimeras and other derivative OB-protein with a long half-life, to the containing compositions and to methods of their introduction. In addition, the invention relates to a method of treating obesity by introducing variants of OB-protein with a long half-life, such as OB-protein-immunoglobulin chimeras.

Air the pits affects about 1/3 of all Americans aged 20 years or older (Kuczmarski et al. , J. Am. Med. Assoc. 272, 205-11 (1994)). Obesity is responsible for a number of serious problems with health, including cardiovascular disorders, type II diabetes, insulin resistance, hypertension, hypertriglyceridemia, disporportionality, and some forms of malignant neoplasms (Pi-Sunyer, F. X. , Anns. Int. Med. 119, 655-60 (1993); Colfitz, G. A. , Am. J. Clin. Nutr. 55, 503S - 507S (1992)). It is shown that the mutation adenocarinoma gene (obesity or ob mutation leads to obesity and type II diabetes in mice (Friedman, Genomics 11, 1054-1062 (1991)). In the work of Zhang et al. Nature 372, 425-431 (1994) recently reported the cloning and sequencing of the ob gene in mice and its homologue related to man, and it was suggested that the product of the ob gene can function as part of a signaling pathway from adipose tissue, which affects the regulation of the magnitude of the stock of fat in the body. Research namely parabiosis spent more than 20 years ago, predicted that the mouse is genetically suffering from obesity, containing two mutant copies of the ob gene (ob/ob mouse), does not produce satiety factor that regulates food consumption, while the mouse suffering from diabetes (db/db), produces a satiety factor, but does not react to it (Coleman, Hummal; Am. J. Physiol. 217, 1298-1304 (1969); Coleman, Diabetol 9, 294-98 (1973)). Recent reports of three based) OB protein inhibit food intake and reduce body weight and fat deposition in ob/ob mice, suffering from excessive obesity, but not in db/db mice (Pelleymounter et al. , Science 269, 540-43 (1995); Halaas et al. , Science 269, 543-46 (1995); Campfield et al. , Science 269, 546-49 (1995)), suggesting that the ob protein is a factor of fullness (satiety), as has previously been assumed in studies on cross-circulation. The results of these first studies left many unresolved questions and showed a number of still unresolved contradictions. For example, although moderate the effects of daily injections ob protein on food intake and body weight were reported for lean mice, there was a significant decrease of reserve fat in the body due to body structure (composition) in one (Halaas et al. , supra), but not in another (Pelleymounter et al. , supra) of these messages, despite an equivalent reduction of body weight. In addition, Pelleymounter et al. , supra noticed that for unknown reasons in the treatment of ob/ob mice a dose of 0.1 mg/kg/day OB-protein really increased their body weight on 17,13%, while the decrease in the weight of mice suffering from obesity who received a dose of ob 1 mg/kg/day was rather small. The receptor or ob receptor protein is still not identified. Although the existence of peripheral receptors cannot currently be excluded, the last message is Adamu phenotype, suggests that OB protein does not act directly on fat cells (Maffei et al. , Proc Natl. Acad. Sd. 92, 6957-60 (1995)). Scientists suggest that at least one OB receptor located in the brain. About identification and expression cloning latinoware receptor (OB-R) was reported in the work Tartaglia et al. Cell 83, 1263-71 (1995). Different isoforms latinoware receptor described Ciofi et al. , Nature Medicine 2, 585-89 (1996). Hematopoietin receptor human, which can be OB receptor protein described in PCT application Publication No. WO 96/08510, published 21 March 1996). The OB receptor, a protein found Tartaglia et al. Cell 83, 1263-71 (1995).

The present invention is based on the observation that OB protein is much more effective to reduce the body weight and the weight of adipose tissue, if you enter a continuous subcutaneous infusion, than if the same dose is given daily subcutaneous injection. In addition, the invention is based on unexpected data, which chimeric protein, in which the OB polypeptide fused to a constant region of an immunoglobulin, is much more effective to reduce body weight and fat than native OB man, if both proteins are injected subcutaneous injection once a day. The last observation is particularly unexpected, so the AI to overcome the blood-brain barrier and reach the Ob receptor, which, I suppose, is located in the brain.

In one embodiment, the invention relates to a derivative of S-protein with a long half-life, capable of reducing body weight and/or food consumption in treated people. In addition, the invention relates to compositions containing such derivatives and introducing them to reduce body weight and/or food intake.

In another embodiment, the invention relates to chimeric polypeptides containing the amino acid sequence of S-protein capable of associating with native OB-receptor attached to the sequence of immunoglobulin (for brevity, called OB-immunoglobulin chimeras or immunoadhesins). In particular (specific) variant chimeric polypeptides include fusion OB-amino acid sequence capable of binding native S-receptor, with a sequence of constant region of immunoglobulin. Preferably, OB-region chimeras described in this invention, would have a sufficient number of amino acid sequences of the native S-protein to preserve its ability to bind to native OB-receptor and signal through a native S-prescriptions the th suffering from obesity people or entities, not related to the human race. OB polypeptide preferably refers to a person, and the merger takes place preferably with a sequence of a constant region of the heavy chain of immunoglobulin. In a separate (private) version, the Association of the two mergers heavy chain OB-polypeptide - immunoglobulin (e.g., by covalent binding using disulfide(s) connection(s)) leads to homodimeric immunoglobulin-like structure. Light chain of immunoglobulin, in addition, you may contact one or both OB-immunoglobulin chimeras in dimer connected by disulfide bonds, with the formation of homotrimers or homotetrameric patterns.

In addition, the invention relates to nucleic acid that encodes a chain chimeric (hybrid) polypeptide, described in this invention, expressing the vectors containing DNA encoding such molecules, transformed host cells and to methods for producing molecules by culturing the transformed cells of the host.

Despite the fact that the derivatives with long half-life, described in this invention, especially suitable for reducing body weight and/or eating, they are just the OB gene and/or to establish (evocation) biological response, called OB receptor. Thus, OB-derivatives described in this invention can be used for the treatment of bulimia, to reduce insulin levels, for example, in patients with I and II types of diabetes and as mitogens different types of cells expressing OB-receptor. All these and other applications are within the scope of this invention.

In another embodiment, this invention relates to a cleaning OB-receptor using OB-protein-immunoglobulin chimeras.

Fig. 1. The top drawing (Fig. 1A). Thin female mice were treated related to mice or rats OB protein using a continuous subcutaneous infusion or daily subcutaneous injection. The data presented are the average body weight of each group in grams, n= 4 mice/point.

Fig. 1. The lower figure (Fig. 1B). Shows the average weight of retroperitoneal fatty. Continuous subcutaneous infusion (infusion) OB protein is also more effective than daily subcutaneous injections for weight reducing adipose tissue.

Fig. 2. The top drawing (Fig. 2A). Obese ob/ob mice were treated OB protein human (hOB), or OB-IgG-1-hybrid protein-related people the dust from the first to the last day of the experiment, in grams, n= 3 mice/column chart, with the exception of injection hOB 0.19 mg/kg/day, where n= 4 and the injection of PBS (phosphate buffered saline), where n= 1.

Fig. 2. The bottom drawing (Fig. 2B). The data presented were the average food intake for each treatment group for the six 24-hour periods of the experiment, in g/mouse/day, n= 1/column charts.

Fig. 3. The upper and lower drawings (Fig. 3A and Fig. 3B). Obese female ob/ob mice were treated or OB protein human (hOB) or hOB-IgG-1-hybrid protein belonging to the person using daily subcutaneous injection for 7 days. Data are depicted as in Fig. 2, with n= 4 for all treated groups.

Fig. 4. The top drawing (Fig. 4A). Obese female ob/ob mice were treated OB protein human (hOB) or PEG-hOB. The data presented are the average changes in body weight for each treatment group from the first to the last day of the experiment, in grams, n= 3-4 mice/column charts, except for the injection of phosphate buffered saline solution, where n= 1. The substances were injected daily subcutaneously. "PEG IX" and "PEG 2X" refers to the ratio of PEG reagent (peg) to a protein when the floor is their food for each treatment group for the six 24 hour periods of the experiment, g/mouse/day, n= 3-4 mice/column charts.

Fig. 5. Obese (ob/ob) female mice were treated or hOB-IgG-treated hybrid protein, the native OB protein human (hOB), or hCD4-IgG using daily subcutaneous injection for 7 days, n= 6 for all treatment groups, except for hOB at 3.8 mg/kg/day, where n= 2. Again observed that the hybrid protein was more effective for reducing weight of the body (upper and middle drawings - Fig. 5A and Fig. 5B) and food intake (lower drawing of Fig. 5B) than native hOB protein.

Fig. 6. The nucleotide sequence (SEQ. ID. No: 1) and amino acid sequence (SEQ. ID. No: 2) OB-IgG-1 chimeras of example 1 relating to the person.

A. Definition

The term "obesity" is used to denote a condition associated with obesity, excessive body fatness. Suitable weight for a particular individual depends on a number of factors, including gender, height, age, overall body shape, and so on, the same factors will be determined, suffer if the individual is obese. Determination of the optimal body weight of the individual is within the competence of the ordinary doctor.

The phrase "long-life" and its grammatical the half-life of plasma and/or slower clearance, than the corresponding native OB-protein. Preferably, derivatives with a long half-life would have a half-life of at least about 1.5 times longer than the native OB protein; more preferably at least about 2 times longer than the native OB-protein; and most preferably at least about 3 times longer than the native OB-protein. Native OB protein is preferably a protein of the individual, which are treated.

The term "OB", "S-polypeptide", "S-protein and its grammatical variants are used interchangeably and refer to a native OB-proteins or "native sequence" OB protein (also known as "leptin") and their functional derivatives. OB polypeptides have the typical structural properties of cytokines, i.e., polypeptides that are released by a single population of cells, which act on another cell as intercellular mediators, such as growth hormone, insulin-like growth factors, interleukins, insulin, glycoprotein hormones such as follicle-stimulating hormone follicle-stimulating hormone. FSH), thyroid-stimulating [thyrostimulin] as NGF-, PDGF; transforming growth factors (transforming growth factors, TGFs) such as TGF - and TGF-, insulin-like rostovy factor-1 and -2 (insulinlike growth factor-1 and -2, IGF-1 and IGF-2), erythropoietin, osteoinductive factors, interferons (interferons, IFNs), for example, IFN-, IFN-, and IFN-; colony stimulating factors (colony-stimulating factors, CSFs) such as M-CSF, GM-CSF and G-CSF; interleukins (interleukins, ILs)for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 and other polypeptide factors.

The terms "native" OB polypeptide or "native sequence" OB polypeptide are used to indicate the OB polypeptide from any species of animal (e.g., related to human, mouse or rat, rabbit, cat, cow, sheep, chicken, pig, horse and so on ), as found in nature, including naturally occurring variants, alleles, deletions, substitutions and/or insertions that are currently known or that may be identified in the future, provided they will retain the ability to bind with OB receptor and preferably will give a signal by OB-receptor. Thus, native OB polypeptide person includes the amino acid sequence between the N-terminal sequence and cysteine (Cys) at position 167 amino acid sequence presented is, currently known or can be identified in the future. Similarly, native OB polypeptide or "native sequence" OB polypeptide related to mice and rats, has the amino acid sequence shown in Fig. 6 from the work of Zhang et al. , supra, and naturally occurring variants of the polypeptide that are currently known or that may be identified in the future. The definition specifically excludes options with glutamine at position 49 or without using the numbering of the amino acids from Zhang et al. , supra. The terms "native" OB polypeptide and a "native sequence" OB polypeptide include native proteins with the initiating N-terminal methionine (Met) or without it and with the native signal sequence or without it, or in Monomeric or dimeric form. Famous native OB polypeptides related to human and mice or rats, have a 167 amino acids contain two saved cysteine residue and have the properties (features) secreted (secreted) protein. Polypeptides substantially hydrophilic, and the predicted cleavage site of the signal sequence is in position 21 using the numbering aminocell or rats, approximately 84%. The two proteins show a broader identity in the N-terminal region of the Mature protein with only four conservative and three non-conservative substitutions among residues between the site of cleavage of the signal sequence and stored Cys at position 117. Molecular weight of OB protein in Monomeric form is about 16 kD.

"Functional derivatives" of the native polypeptide is a compound that has a qualitative biological property (ability), in common with the native polypeptide. Functional derivatives OB polypeptide is a compound that has a qualitative biological property in common with the native OB polypeptide (and non-human race). "Functional derivatives" include, but are not limited to fragments of the native polypeptides from any species of animals (including humans) and derived native polypeptides and their fragments (and non-human race), provided that they have a biological activity in common with the corresponding native polypeptide.

"Fragments" include the area within the sequence of the Mature native OB-polypeptidic contain relatively short(s), deletion(s) at the N-terminal sequence and in other parts of the molecule, unused to bind the receptor and/or structural integrity.

The term "derivative" is used to define amino acid sequence variants and covalent modifications of a native polypeptide, whereas the term "variant" refers to amino acid sequence variants within this definition.

"Biological property" in the context of the definition of "functional derivatives" is defined either as 1) immunological cross-reactivity with at least one epitope (antigenic determinant) of the native polypeptide (e.g., native OB polypeptide of any kind), or 2) having at least one adhesive, regulatory or effector function qualitatively in common with the native polypeptide.

Preferably, the functional derivative was polypeptides which have at least 65% amino acid sequence identity, more preferably about 75% amino acid sequence identity and even more preferably at least about 85% amino acid sequence identity and most preferably at least about 95% of identichnost derived native sequence OB polypeptide, related to the human race, preferably show at least 95% identity with amino acid sequence of native OB-proteins and are not immunogenic in humans.

Identity or gomologichnosti amino acid sequence of the present invention is defined as the percentage of amino acid residues in the sequence of candidate residues which are identical to the corresponding sequence of the native polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, and without considering any conservative substitutions as part of the identity sequence. Either N - or C-terminal extension segments or insertional segments should not be considered as reducing identity or gomologichnosti.

Immunologica cross-reactive, as it is used in this invention, means that the (poly)peptide of the candidate is capable of competitively inhibiting the qualitative biological activity of a corresponding native polypeptide having this activity with polyclonal antibody or an antiserum directed against Islam introduction, for example, goat or rabbit subcutaneously with known native OB protein complete adjuvant's adjuvant, followed by booster injection intraperitoneal or subcutaneous injection incomplete adjuvant-blockers.

The term "isolated" (isolated) OB polypeptide" and its grammatical variants belong to OB-polypeptides (as defined above), separated from contaminant polypeptides present in man, other animals, or other source from which the polypeptide is selected.

Usually the term "variant amino acid sequences" refers to molecules with some differences in their amino acid sequences compared to the original (for example, the native sequence) polypeptide. Changes of amino acids can be made by substitutions, insertions, deletions or any necessary combination of such changes in the native amino acid sequence.

Variants of substitution (replacement) are those which have at least one removed amino acid residue in the native sequence and the introduction instead of the other amino acids in the same position. The substitution can be only replaced if only one ammeyah in the same molecule.

Insertional variants are variants with one or more amino acids introduced directly adjacent to an amino acid at a certain position in the native amino acid sequence. Directly next door to the amino acid means linking or p-carboxy - or amino-functional group of amino acids.

Options deletions are variants with one or more of the deleted amino acids in the native amino acid sequence. Usually options deletions should have one or two amino acids subjected to deletions in a specific region of the molecule.

"Covalent derivatives" include modification of the native polypeptide or its fragment organic protein or non-protein derivatization agent, and posttranslational modification. Covalent modifications are traditionally introduced by reacting certain amino acid residues with an organic derivatizing agent, which is capable of reacting with selected sites or terminal residues, or by using mechanisms "pragana" post-translational modifications that function in selected recombinant cells of the host. Some posttranslational modification I have asparaginamide often deleteroute excision to the corresponding glutamyl and esportillada. Or these residues deleteroute in moderately acidic conditions. Every form of these residues may be present in OB-immunoglobulin the chimeras described in this invention. Other post-translational modifications include hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups merilnyh, tyrosine or traveling residues, methylation of the amino groups of the side chains of lysine, arginine and histidine (I.e., Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co. , San Francisco, pp. 79-86 (1983)).

The term "DNA sequence encoding," and "DNA encoding" and "nucleic acid encoding" refer to the order or sequence deoxyribonucleotides along the chain deoxyribonucleic acid. The sequence of these deoxyribonucleotides determines the order of amino acids along the polypeptide chain. Thus, the DNA sequence encodes the amino acid sequence.

The term "able to replicate expressing vector and expressing the vector" refers to a fragment of DNA, usually double-stranded, which may be given in a fragment of another (foreign) DNA. Foreign DNA is defined as the heterologous DNA, which is DNA not found in klemke host, the vector may replicate independently of the chromosomal DNA of the host, you can generate multiple copies of the vector and the inserted foreign DNA with alien DNA insert). In addition, the vector contains the necessary elements that allow to transform someone else's DNA in the polypeptide. Thus, it can quickly be synthesized many of the molecules of the polypeptide encoded by foreign DNA.

The term "control sequences" refers to DNA sequences necessary for expression directly (resectable) linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, an operator may plot (sequence), ribosom.okazavaet site and possibly others still poorly understood sequence. It is known that eukaryotic cells use promoters, polyadenylated signals and enhancer.

Nucleic acid is directly (resectable) connected", if it is placed in a functional relationship from the other nucleic acid sequence. For example, DNA - predpolagavshegosja or secretory leader sequence operable connected with DNA polypeptide, if he expresses ka(resectable) is connected to the coding sequence, if it affects the transcription of the sequence; or ribosom.okazavaet the website is directly linked to the coding sequence if it is so easy translation. Usually directly (resectable) coupled" means that the DNA sequences being United, are close, and in the case of a secretory leader sequence are close and in the phase of reading. However, the enhancers should not be close. The connection is achieved by legacies in a convenient restriction sites. If such sites do not exist, then, in accordance with the accepted practice of using synthetic oligonucleotide adaptors or linkers.

In the context of this invention the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells (host)" include the primary subject cell and cultures derived from them, not taking into account the number of transfers. You should also understand that all offspring should not be completely identical in DNA content, due to deliberate or inadvertent mutations. Included mutant offspring that ilayh cells. Where are assumed to be perfect symbols will be clear from the context.

Native immunoglobulins are usually heterotetrameric glycoproteins with a molecular weight of about 150,000 daltons, composed of two identical light (light (L) chains and two identical heavy (heavy, N) circuits. Each light chain is linked to a heavy chain through covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has a naturally located inside the chain disulfide bridges. Each heavy chain has at one end of the variable region (VH), followed by a number of constant domains. Each light chain has a variable region (VL) and one constant region at the other end; the constant region of the light chain is in line with the first constant region of the heavy chain and the variable region of the light chain is on the same line as the variable region of the heavy chain. Believe that certain amino acid residues form a boundary surface between the variable regions of the light and heavy chains (Clothia et al. , J. Mol. Biol. 186. 651-663 (1985); Novotny, Haber, Proc. Natl. Acad. Sci. USA 82, 4592-4 their heavy chains belong to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can further be divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. Constant region heavy chains, which correspond to the different classes of immunoglobulins are called, , , , , respectively. Well-known structures of subunits and three-dimensional configurations of different classes of immunoglobulins. IgA-1 and IgA-2 are Monomeric IgA subclasses, which is usually in the form of dimers or polymers with a large number of monomers. The immune cells in the gut produce mainly of polymeric IgA (also applies to poly-IgA, including dimers and polymers with a large number of monomers). Such poly-IgA contains linked by a disulfide bridge polypeptide, called "connecting" peptide or "J-peptide, and can be transported through glandular epithelium together with J-containing polymeric IgM (poly-IgM), comprising five subunits.

Hybridization is preferably carried out at the "exact circumstances", which means (1) the use of solutions with low ionic strength and high temperature for washing, for example of 0.015 M sodium chloride/0,0015 M sodium citrate/0.1% sodium dodecyl sulphate at 50oC. another example is use of 50% of formamide, 5xSSC (0.75 M NaCl, Of 0.075 M sodium citrate), 50 mm sodium phosphate (pH 6/8), 0.1% sodium pyrophosphate, 5 x denhardt's solution (Denhardt''s solution), the homogenate obtained from cells DNA salmon sperm when exposed to ultrasound (50 µg/ml), 0.1% of SDS (sodium dodecyl sulphate) and 10% dextran sulfate at 42oC, with washes at 42oC 0.2 xSSC and 0.1% SDS.

C. OB-protein-immunoglobulin chimeras (immunoadhesin)

Immunoadhesin are molecules similar to the chimeric antibody, which combine functional(s) region(s) binding protein (usually a receptor, a molecule, or a ligand cell adhesion) with the sequence of the immunoglobulin. The most common example of this type of hybrid protein combines the hinge and Fc region of immunoglobulin (Ig) domains (regions) of the cell-surface receptor that recognizes a specific ligand. This type of molecule is called a "immunoadhesin", because it combines "immune" and "adhesive" function; another common name is "Ig Chimera", "Ig" or "Fc-hybrid protein" or "receptor globulin".

At present, there are over fifty immunoadhesins. And the l. , Proc. Natl. Acad. Sci. USA 84, 2936-2940 (1987)); CD4 (Capon et al. Nature 337, 525-531 (1989); Traunecker et al. , Nature 339, 68-70 (1989); Zettmeissi et al. , DNA Cell Biol. USA 9, 347-353 (1990); Bym et al. , Nature 344, 667-670 (1990)); L-selectin ("homing"-receptor) (Watson et al. , J. Cell. Biol. 110, 2221-2229 (1990); Watson et al. , Nature 349, 164-167 (1991)); E-selectin (Mulligan et al. , J. Immunol. 151, 6410-17 (1993); Jacob et al. , Biochemistry 34, 1210-1217 (1995)); P-selectin (Mulligan et al. , supra; Hollenbaugh et al. , Biochemistry 34, 5678-84 (1995)); ICAM-1 (Stauton et al. , J. Exp. Med. 176, 1471-1476 (1992); Martin et al. , J. Virol. 67, 3561-68 (1993); Roep et al. , Lancet 343, 1590-93 (1994)); ICAM-2 (Damle et al. , J. Immunol. 148, 665-71 (1992)); ICAM-3 (Holness et al. , J. Biol. Chem. 270, 877-84 (1995)); LFA-3 (Kanner et al. J. Immunol. 148, 2-23-29 (1992)); L1 glycoprotein (Doherty et al. , Neuron 14, 57-66 (1995)); TNF-R1 (Ashkenazi et al. , Proc. Natl. Acad. Sci. USA 88, 10535-539 (1991); Lesslauer et al. , Eur. J. Immunol. 21, 2883-86 (1991); Peppel et al. , J. Exp. Med. 174, 1483-1489 (1991)); TNF-R2 (Zack et al. , Proc. Natl. Acad. Sci. USA 90, 2335-39 (1993); Wooley et al. , J. Immunol. 151, 6602-07 (1993)); CD44 (Aruffo et al. Cell 61, 1303-1313 (1990)); CD28 and B7 (Linsley et al. , J. Exp. Med. 173, 721-730 (1991)); CTLA-4 (Lisley et al. , J. Exp. Med. 174, 561-569 (1991)); CD22 (Stamenkovic et al. Cell 66, 1133-1144 (1991)); NP receptors (Bennett et al. , J Biol. Chem. 266, 23060-23067 (1991)); IgE receptor (Ridgway and Gorman, J, Cell. Biol. 115, abstr. 1448 (1991)); HGF receptor (Mark, M. R. et al. , 1992, J. Biol. Chem. submitted)); IFN-R - and-chain (Marsters et al. , Proc. Nail. Acad. Sci. USA 92, 5401-05 (1995)); trk-A-b-C (Shelton et al. , J. Neuriosci. 15, 477-91 (1995)); IL-2 (Landolfi, J. Immunol. 146. 915-19 (1991)); IL-10 (Zheng et al. , J. Immunol. 154, 5590-5600 (1995)).

The simplest and most direct design immunoadhesin byebye, when you get OB-immunoglobulin chimeras described in this invention, nucleic acid encoding the desired OB polypeptide, should merge the C-end of the nucleic acid that encodes a N-terminal sequence of the constant region of the immunoglobulin, however N-terminal fusion is also possible. Usually, when such mergers encoded chimeric polypeptide will retain at least functionally active hinge, CH2 and CH3 domains of the constant region of the heavy chain of immunoglobulin. Mergers are also held with C-end Fc area constant area or directly to the N-Terminus with CH1 of the heavy chain or the corresponding region of the light chain. The exact site on which there is a merger, not a critical; particular sites are well known and can be selected in order to optimize the biological activity, secretion or binding characteristics of the OB-immunoglobulin chimeras.

In a preferred embodiment, the sequence of native Mature OB polypeptide is fused to the N-end C-end portion of an antibody (in particular the Fc domain), containing the effector functions of an immunoglobulin, such as IgG-1. It is possible to drain all the constant region of the heavy chain with OB-sequence the region, just in reverse (3'-5' direction) from the cleavage site of papain (which defines IgG Fc chemically; residue 216, taking the first residue of the constant region of the heavy chain - 114 (Kobet et al. , supra), or analogous sites of other immunoglobulins). In a particularly preferred embodiment, the sequence of OB polypeptide is fused to the hinge region and CH2 and CH3 or CH1, hinge, CH2 and CH3 regions of the heavy chain of IgG-1, IgG-2 or IgG-3. The exact site where there is a merger, not a decisive and optimal site can be determined by routine experimentation.

In some embodiments, OB-immunoglobulin chimeras are multimeric and, in particular, in homodimer or tetramer (WO 9108298). Typically, these collected immunoglobulins consist of known isolated structures. The main chetyrekhzvezdochnoj structural unit is the form in which there are IgG, IgD and IgE. Chetyrekhzvezdochnaya unit is repeated in immunoglobulins larger molecular weight; IgM usually exists in the form of pentamer main chetyrehzaryadnyh units, interconnected by disulfide bonds. IgA globulin and sometimes IgG globulin may also exist in multimeric form in serum. In the case of multimer each chetyrehzvennoy chimeras within the scope of the invention are represented schematically below:

(a) ACL-ACL;

(b) AUH- [°CHACL-ASHACL-VHWITHHor VLWITHL-ACH] ;

(C) AUL-ASH-[ACL-ASHACL-VHWITHHVLCL-ASHor VLCL-VHCH] ;

(g) AUL-VHCH- [°CHor AUL-VHCHor VLCL-ACH] ;

(d) VLWITHL-ASH- [°CL-VH-CHor VLCL-ASH] , and

(e) [A-Y]n[VL-CL-VHWITHH] 2,

where And each represents the same or different amino acid sequence OB polypeptide;

VL- variable region light chain immunoglobulin;

VH- variable region of the heavy chain of immunoglobulin:

WITHL- constant region light chain immunoglobulin;

WITHH- constant region of the heavy chain immunoglobulin;

n is an integer greater than 1;

Y denotes a residue cross-linking (covalent) agent.

In the interests of brevity, the above patterns show only the basic properties; they do not indicate connection (J) or other areas of Amnesty, they must be created, and must be defined in the usual places that they occupy in the molecules of immunoglobulins.

On the contrary, OB-amino acid sequence can be inserted (to fit) between the sequences of the heavy chain and light chain immunoglobulin, so get an immunoglobulin comprising a chimeric heavy chain. In this embodiment OB polypeptide sequence fused to the 3'end of the heavy chain of immunoglobulin with each arm of an immunoglobulin, or between the hinge and CH2 regions, or between the CH2 and CH3 regions. About similar structures were reported Hoogenboom, H. R. et al. , Mol. Immunol. 28, 1027-1037 (1991).

Although the presence of the light chain of immunoglobulin is not required in immunoadhesin described in this invention, the light chain immunoglobulin may be present or kovalentnosvyazannoi with OB protein-immunoglobulin heavy chain hybrid polypeptide, or directly to merge with OB polypeptide. In the former case, DNA encoding a light chain immunoglobulin, usually coexpressed with DNA encoding OB-immunoglobulin heavy chain hybrid protein. When secretion of the heavy chain and light chain of the hybrid will be covalently contact, giving alpinum bridge. The method is suitable for obtaining such structures, for example, was proposed in a U.S. patent (U. S. Patent No. 4816567, issued 28 March 1989).

In a preferred embodiment, the sequence of the immunoglobulin used in the creation of immunoadhesins described in the present invention, represent the constant region of the heavy chain IgG immunoglobulin. For immunoadhesins person it is preferable to use a sequence of IgG-1 and IgG-3 antibodies, specific to the person. The main advantage of using IgG-1 is that IgG-1 immunoadhesin can be easily cleaned on immobilized protein A. on the Contrary, the clearance of IgG-3 requires the G protein - significantly less mobile environment. However, you should consider other structural and functional properties of immunoglobulins, when you choose lg hybrid partner to create this immunoadhesin. For example, IgG-3 hinge region longer and more flexible, so it can fit large "adhesive" region, which can not bend or function properly when fused with IgG-l. Possible patterns immunoadhesin on the basis of IgG are shown in Fig. 3 A-Century Since IgG immunoadhesin are usually mono - or DV is respectively, main Ig homodimeric units. Typical multimeric immunoadhesin on the basis of IgM is shown in Fig. 3, a Multimeric immunoadhesin advantageous in that they can bind their respective targets with greater avidity than their counterparts based on IgG. Examples of such structures are CD4-IgM (Traunecker et al. , supra); ICAM-IgM (Martin et al. , J. Virol. 61, 3561-68 (1993)); and CD2-IgM (Arulanandam et al. , J. Exp. Med. 177, 1439-50 (1993)).

For OB-Ig-immunoadhesins, which are intended for use in vivo, are also important pharmacokinetic properties and the effector functions specified by the Fc region. Although IgG-1, IgG-2, IgG-4 - all have a half-life of 21 days, their relative effectiveness in activation complemental system is different. IgG-4 does not activate complement and IgG-2 significantly weaker when activation of complement than IgG-1. In addition, in contrast to IgG-1, IgG-2 does not bind to Fc receptors on mononuclear cells or neutrophils. Whereas IgG-3 is optimal for the activation of complement, its half-life in vivo is approximately one-third of other IgG isotypes. Another important consideration regarding immunoadhesins intended for use as therapeutic agents for humans, is the number of the allotype is th allotype. For example, IgG-1 has only four typed by anticorodal alltimecase site, two of which (G1m and 2) are located in the Fc region; and one of these sites G1m1 is not immunogenic. On the contrary, there are 12 typed by anticorodal of allotypes in IgG-3, they are all in the Fc-region; only three of these sites (G3m5, 11 and 21) have the same allotype, which is non-immunogenic. Thus, the potential immunogenicity of3immunoadhesin greater than the potential immunogenicity of1immunoadhesin.

Related OB-Ig immunoadhesin described in this invention, the areas that are not required for receptor binding, structural integrity (for example, corresponding folding (styling) and/or biological activity of the molecule, can be erased. In such structures it is important to place the junction of the merger on the remains, which are located between the areas (domains) in order to avoid erroneous folding. With regard to the parent immunoglobulin, need a point of junction is in reverse (3'-5' direction) from cysteine hinge region, which form a disulfide bond between the two heavy chains. In a frequently used design, the codon C-terminal residue "adhesins is lnasty IgG-1 hinge region.

OB-Ig-immunoadhesins are most conveniently through the merger of the cDNA sequence that encodes OB-the area within the reading frame, with Ig cDNA sequence. However, it can also be used to merge with genomic Ig fragments (see, for example, Gascoigne et al. , Proc. Natl. Acad. Sci. USA 84, 2936-2940 (1987): Aruffo et al. Cell 61, 1303-1313 (1990); Stamenkovic et al. Cell 66, 1133-1144 (1991)). The latter type of fusion requires the presence of Ig regulatory sequences for expression. cDNA-you're encoding the constant region of IgG heavy chains, can be isolated based on published sequences from cDNA libraries derived from spleen and peripheral blood lymphocytes using a hybridization method or the method of polymerase reaction synthesis circuit (polymerase chain reaction, PCR). OB cDNA related to mice and rats, it can, for example, be obtained using the polymerase synthesis reaction chain from cDNA libraries of adipose tissue mouse (Clontech) using primers designed on the basis of sequence Zhang et al. Similarly, you can get OB cDNA specific for the human race. Or you can use OB mouse gene as a probe to isolate cDNA clones adipose tissues (Clontech), for example from gtll library, as described in Zhang et al. KD directs efficient expression in the chosen host cell. For expression in mammalian cells is preferred vector based on pRK5 (Schall et al. Cell 61, 361-370 (1990)), pRK7-vector and vector-based CDM8 (Seed, Nature 329, 840 (1989)). (pRK7 identical pRK5, except that the order (sequence) sites of restriction endonucleases in polylinker region between the ClaI and HindIII opposite (see U. S. Patent No. 5108901? issued 28 April 1992). The exact scope of the connection can be created by removing an additional sequence between the created codons connection using site-directed mutagenesis using the oligonucleotide (Zoller, Smith, Nucleic Acids Res. 10, 6487 (1982); Capon et al. Nature 337, 525-531 (1989)). Can be used synthetic oligonucleotides, in which each half of the complementary sequences on each side of the desired area connection: ideally they range from 36 - to 48-mers. Or the polymerase reaction synthesis circuit can be used to connect two parts of the molecule inside the reading frame with the corresponding vector.

Immunoadhesin can effectively Express in various host cells, including myeloma cell line, cells of the ovary of the Chinese hamster (Chinese Hamster ovary, CHO), COS cells monkeys, 293 embryonic kidney cells is chosen and secrete (highlighted) in the cell culture medium. As hosts can also be used in yeast, for example Saccharomyces cerevisiae, Pichia pastoris, etc., and bacterial cells, preferably E. coli. OB-immunoglobulin chimeras can Express in yeast, for example, analogously to the process described for the expression of OB protein in the works Leiber et al. Crit. Res. Food Sci. Nutr. 33, 351 (1993); Friedman, Leibel, Cell 69, 217 (1992); and Beavis, Chait, Proc. Natl. Acad. Sci. USA 87, 6873 (1990). Thus, the coding sequences can subconious in yeast plasmid, such as a plasmid pPIC g(Invitrogen) expression of the yeast. This vector directs secretion of heterologous proteins from yeast in the medium for cultivation. According Halaas et al. , supra expression of the OB-gene mouse and human rights in Saccharomyces cerevisiae transformed data vector, leads to the production of secreted 16-kD protein, which is reprezentirovanii OB protein without the signal sequence. The expression of OB-immunoglobulin chimeras of the mouse and human rights in E. coli may, for example, by analogy with the method described Halaas et al. , supra. Coding sequences OB-immunoglobulin chimeras of the mouse and human can subconious in 15b expressing vector (Novagen) and expressed in E. coli (BL21 (DE3)plYsS) when ispolzovaniya sequence into the reading frame sequence of the secretion of thermostable enterotoxin II E. coli, in the forward direction (5'-3' direction) of the promoter of E. coli alkaline phosphatase (Chang et al. Gene 55, 189-96 (1987)).

The line selection of host cells for the expression of OB-Ig-immunoadhesins depends mainly on expressing vector. Another consideration is the amount of the desired protein. Milligramme quantity often can be obtained by means of temporal transfection. For example, adenovirus EIA-transformed 293 embryonic kidney cells human can temporarily transfectants with pRK-5 and pRK7 vector by modifying the calcium phosphate method to achieve efficient expression immunoadhesin. This method is illustrated by examples. Vectors based on the CDM8 can be used for transfection of COS cells DEAE-dextranomer method (Aruffo et al. Cell 61, 1303-1313 (1990); Zettmeissl et al. , DNA Cell Biol. (US) 9, 347-353 (1990)). If you need large quantities of protein, immunoadhesin can be expressed after stable transfection cell line owner. For example, vector-based or pRK5 pRK7, may be introduced into the cells of the Chinese hamster ovary (CHO) in the presence of an additional plasmid encoding dihydrotetrazolo (dihydrofolate reductase, DHFR) and imparts resistance to G418. Clones resistant (resistant to G418 - is nhibitor digidrofolatreduktazy; clones selectyou, in which the number of copies of genes coding for the DHFR sequence and immunoadhesin together, it provided amplification. If immunoadhesin contains a hydrophobic leader sequence at its N end, then most likely it will processional and secrete transfecting cells.

Expression of immunoadhesins more complex structures may require uniquely appropriate host cells, for example, components of the light chain or "J" peptide can be provided by some myeloma or hybridoma cell owners (Gascoigne et al. , 1987, supra; Martin et al. J. Virol. 67, 3561-3568 (1993)). Expression of immunoadhesins with more complex oligomeric structures may require uniquely appropriate host cells, such as the components of the light chain or "J"-peptide can be provided by some myeloma or hybridoma cell owners (Gascoigne et al. ,

supra, Martin et al. , J. Immunol. 61, 3561-68 (1993)).

Immunoadhesin can easily be purified using affinity chromatography. The suitability of protein And as an affinity ligand depends on the species and isotype Fc region of immunoglobulin, which is used in the Chimera. Protein And can be used for cleaning Edu (Lindmark et al. , J. Immunol. Meth. 62, 1- 13 (1983)). Protein G is recommended for all isotypes specific to mouse, and3belonging to the human race (Guss et al. , EMBO J. 5, 1567-1575 (1986)). The matrix is an affine ligand, is most frequently the agarose, but other suitable matrix. A mechanically stable matrices such as glass with a defined pore size or poly(Stradivari)benzene, allow to increase the flow velocity and reduce processing times than those achieved with agarose. Bonding conditions immunoadhesin with protein a or G affinity column is completely dictated by the characteristics of the Fc-region; i.e., species and isotype. Usually, when you select the appropriate ligand is effective linking directly from absolute cultural liquid. One of the distinguishing characteristics of immunoadhesins is that1molecules belonging to the human race, the binding ability of the protein And is somewhat reduced in comparison with the antibody of the same Fc type. Associated immunoadhesin can effectively buyouts or at acidic pH (pH 3.0 or above), or in a buffer with neutral pH, containing moderately chaotropic salt. This stage affinity chromatography may lead the person can be used instead of or in addition to affinity chromatography on protein a or P. Immunoadhesin behave in a manner similar to antibodies in teofilina gel chromatography (Hutchens, Porath. , Anal. Biochem. 1596217-226 (1986)) and immobilizovannoi metal complex chromatography (Al-Mashikhi, Makai, J. Dairy Sci. 71, 1756-1763 (1988)). In contrast to antibodies, their behavior on ion-exchange columns is dictated, however, not only their isoelectric points, but also charge dipole that can exist in molecules due to their chimeric nature. Microheterogeneity charge may also be a factor for immunoadhesins in which adhesiva part of the molecule glycosylases and contains sialic acid. The specific procedure of purification described in the examples.

Results from numerous immunoadhesins obtained so far show that the merger adhesieve plot with the Fc region is usually not interfere with the folding of individual domains. As adhesiva and immunoglobulin region seems to fold correctly and Fc region retains many of the effector functions, which are the characteristics of the antibodies, such as binding to Fc receptors.

The methods usually used for the creation, expression and purification of immunoadhesins described, for example, in U.S. patents (U. S. P is OK. Creating immunoadhesin, expression, purification and different patterns of immunoadhesins also described in review articles (Ashkenazi, Chamnov, Methods in Enzymology 8, 104-115 (1995) and Peach, Linsley, Methods in Enzymology 8, 116-123 (1995)), the detection of which along with the references cited, as specifically shown as a reference.

C. Other OB-derivatives with a long half-life.

Other derivatives OB-proteins, which have a longer half-life than the native molecules, contain OB protein or OB-immunoglobulin Chimera covalently-linked with non-protein polymer. Non-protein polymer is typically a hydrophilic synthetic polymer, i.e. the polymer is not found in nature. However, suitable polymers that exist in nature and are produced by recombinant methods or methods in vitro as polymers, which are extracted from natural sources. Hydrophilic polyvinyl polymers, e.g. polyvinylalcohol and polyvinylpyrrolidone are included in the scope of this invention. Particularly suitable polyalkylene ethers such as polyethylene glycol (PEG); polyalkylene, such as polyoxyethylene, polyoxypropylene and block copolymers of polyoxyethylene and polio which which contain carbohydrate monomers D-mannose, D - and L-galactose, fucose. fructose, D-xylose, L-arabinose, D-glucuronic acid, sialic acid, D-galacturonic acid, D-mannurone acid (for example, polymannuronic acid or alginic acid), D-glucosamine, D-galactosamine, D-glucose and neuraminic acid including homopolysaccharides and heteropolysaccharides such as lactose, amylopectin, starch, gidroxiatilkrahmal, amylose, dextran sulfate, dextran, dextrins, glycogen, or the polysaccharide subunit of acid mucopolysaccharides, such as hyaluronate acid; a polymer of carbohydrate-containing alcohols, such as Polysorbate and pelimannit; heparin or heparin. The polymer prior to the introduction of cross-links is not necessarily, but preferably should be soluble, but the final conjugate should be water-soluble. In addition, the polymer should not be highly immunogenic in conjugate form, should not be sticky, which is incompatible with intravenous infusion or injection, if it is intended for such way of introduction.

Preferably, when the polymer contains only one reactive group. This prevents cross-linking of protein molecules. However, within the scope of izopet roducti reaction by gel filtration or molecular sieves for regenerating almost homogeneous derivatives.

The molecular weight of the polymer can be in the range from ~ 100 to 500,000 and preferably be from 1000 to 20000. The choice of molecular weight will depend on the nature of the polymer and the degree of substitution. Generally, the higher the hydrophilicity of the polymer and the higher the degree of substitution, the more likely it is possible to use a polymer with a lower molecular weight. The optimum molecular weight determined by routine experimentation.

The polymer is typically covalently associated with OB protein or OB-immunoglobulinemia chimeras with a multifunctional cross-linking agent that reacts with the polymer and one or more amino acid or carbohydrate residues OB protein or OB-immunoglobulin chimeras can be linked. However, within the scope of the invention also direct cross-linking of the polymer in the interaction derivatizing polymer hybrid or Vice versa.

The site of covalent cross-stitching on the OB protein or OB-Ig contains N-terminal amino group and the amino group present in the residues of lysine and other amino-, imino-, carboxyl, sulfhydryl, hydroxyl or other hydrophilic groups. The polymer may be covalently to contact NuMega agent. Covalent binding of amino groups is achieved is known in chemistry methods based on the use of reactive groups of the acid chloride cyanuric acid, a carbonyl derivative diimidazole, aldehyde (PEG alkoxide + diethylacetal of bromoacetaldehyde; PEG + dimethyl sulfoxide + acetic anhydride, or PEG chloride + phenoxide 4-hydroxybenzaldehyde, active esters succinimide, activated dithiocarbonate PEG, 2,4,5-tricarbonylchromium or n-nitrophenylphosphate activated PEG). Carboxyl group derivatized when linking with PEG-amine, using carbodiimide.

The polymers are connected with groups of oligosaccharide oxidation using chemical substances, such as metaperiodate, or enzymes, such as glucose or galactosidase (each of which leads to the production of aldehyde derivatives of carbohydrates), followed by reaction with hydrazide or aminoderivatives polymers, in the same way as described in the works Heitzmann et al. , P. N. A. S. 71, 3537-41 (1974), and Bayer et al. , Methods in Enzymology 62, 310 (1979) to obtain labeled with Biotin or Avidya oligosaccharides. In addition, especially useful chemical and enzymatic methods that have previously been used in the x sites for derivatization and thus, the oligosaccharide products are more homogeneous. Deputy oligosaccharides may also modify enzymatic cleavage to remove the sugar, for example, by splitting the neuraminidase prior to derivatization of the polymer.

The polymer should contain a group which reacts directly with amino acid side chains or the N-or C-end of a related polypeptide, or which reacts with multifunctional Poperechnaya agent. Typically, the polymers containing such reactive groups known to obtain the immobilized proteins. In order to use such techniques in this invention, it is necessary to apply a water-soluble polymer, derivationally in the same way as before was used for insoluble polymers used for immobilization of the protein. Particularly suitable activation method methyl-cyan for use in cross-linking of polysaccharides.

"Water soluble", as for the original polymer means that the polymer or its reactive intermediate compound used to bind, is quite soluble in water in order to participate in the derivatization reaction.

The degree of substitution such a polymer will vary depending on the number of reactive sites on the protein, whether the whole protein or its fragment, fused whether protein with a heterologous protein (e.g., OB-immunoglobulin Chimera), molecular weight, hydrophilicity and other characteristics of the polymer and from selected parcels derivatization of this protein. Typically, the conjugate contains from about 1 to 10 polymer molecules, although any heterologous sequence may be replaced by a virtually unlimited number of polymer molecules until the desired activity largely will not be rendered harmful effects. The optimal degree of cross-linkage is easily determined by the experimental matrix in which the change of time, temperature and other reaction conditions, to change the degree of substitution, and then determined the ability of the conjugates to function as desired.

Polymer, such as PEG, perekreschivayutsya using a variety of methods, basically known to covalent modification of protein non-protein polymers, such as PEG. Some of these methods,IDA cyanuric acid leads to a large number of adverse reactions, including cross-linking protein. In addition, it can in particular lead to a possible inactivation of proteins containing sulfhydryl groups. Using carbonyldiimidazole (Beauchamp et al. , Anal. Biochem. 131, 25- 33 (1983)) requires high pH (>8,5), which can inaktivirovanie proteins. In addition, since the intermediate "activated PEG" may react with water, requires a very large molar excess of activated PEG" compared with protein. High concentrations of PEG required when using carbonyldiimidazole also create problems when cleaning as this has a deleterious effect on gel filtration (gel chromatography) and chromatography hydrophilic interaction. In addition, the high concentration of activated PEG can precipitate the protein, the problem is what, in fact, noted above (Davis, U. S. Patent No. 4179337). On the other hand, the use of aldehyde (Royer, U. S. Patent No. 4002531) is more efficient because it only requires a 40-fold molar excess of PEG and 1-2 hours of incubation. However, manganese dioxide, proposed by Royer (Royer) to obtain PEG aldehyde problematic "due to the pronounced tendency of PEG to form complexes with the oxidizing agent on the basis of the sulfoxide (DMSO) and acetic anhydride, fixes this problem. In addition, sodium borohydride, proposed by Royer (Royer), to be used at high pH and has a significant tendency to restore the disulfide bonds. On the contrary, the preferred cyanoborohydride sodium, which is effective at neutral pH, and has a very low tendency to restore the disulfide bonds.

PEG polymers with functional groups for modification of the OB protein or OB-Ig-chimeras described in this invention, are available from Shearwater Polymers, Inc. (Huntsville, AL). Such commercially available PEG derivatives include, but are not limited to, amino-PEG esters of amino-PEG, PEG - hydrazide, PEG-thiol, PEG-succinate, karboksimetilirovaniya PEG, PEG-propionic acid, PEG-amino acids, PEG Succinimidyl, PEG-succinimidylester, Succinimidyl ether carboxymethylamino PEG, succinimidylester PEG; Succinimidyl esters of amino acids, PEG, PEG-oxycarbonate, PEG-nitrophenylarsonic, PEG-TResult, PEG-glycidyloxy ether, PEG-aldehyde, PEG vinylsulfonic, PEG-enigmaloginova acid, PEG-o-pyridyldithio, heterofunctional PEG and PEG-vinyl derivatives, PEG-silanes and PEG-pospolity. Reaction conditions linking these PEG derivatives will vary zaveshchaniye when choosing a PEG derivatives include the necessary binding site (lysine or cysteine), hydrolytic stability and reactivity derivatives, stability, toxicity and antigenicity clutch, suitability for analysis, etc. Specific instructions on how to use any data derived are available from the manufacturer.

Conjugates with long life, described in this invention, are separated from the unreacted starting compounds using gel filtration. Heterologous species conjugates purified from one another in the same way. The polymer can also be water-soluble, as a hydrophilic gel.

The conjugates can also be cleaned using ion-exchange chromatography. The chemistry of many electrophilic activated PEG-s leads to a decrease of the charge of the amino group of PEG-lirovannomu product. Thus, ionoobmennaya chromatography high resolution can be used to separate the free and conjugated proteins and to separate particles with different levels of PEG-helirovanie. In fact, the separation of various particles (for example, containing one or two PEG residues) is also possible due to differences in ionic properties of unreacted amino acids.

G. Application of OB-immunoglobulin chimeras and other what's with the long half-life, described in this invention is suitable for reducing weight and, specifically, for the treatment of obesity and other disorders associated with abnormal expression or function of the OB gene. Our studies show that OB-immunoglobulin chimeras and other OB-derivatives with a long half-life, such as PEG-lirovannye OB, reduce food intake and increase energy consumption being treated animals, and therefore are very effective for reducing weight as fat, and normal subjects. For verification purposes, the molecules can be dissolved in a phosphate buffered saline solution (phosphate-buffered saline. PBS) (pH of 7.4) and administered by intravenous or subcutaneous injection or infusion.

OB-derivatives with prolonged action described in this invention can be used also for treatment of other metabolic disorders, such as diabetes and bulimia. It was shown that OB protein reduces the level of insulin in animals and can be used to reduce excessive levels of insulin in humans. The reduction of insulin levels in obese and non-obese individuals patients (e.g. patients with diabetes I or type II) can restore or improve the sensitivity is to be used for the treatment of diseases of the kidneys, hypertension and dysfunction of the lungs, such as emphysema. OB protein can also cause mitogenic response in receptortargeted tissues, acting as a growth factor for these cells.

Therapeutic compositions described in this invention are obtained when mixing the active ingredient having the desired degree of purity with physiologically acceptable carriers, excipients or stabilizers (Remington''s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilised compositions or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations, and include buffers such as phosphate, citrate and buffers other organic acids; antioxidants including ascorbic acid; low molecular weight (less than 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols, as napie substances, such as twin, pluronic or PEG.

The active ingredients can also be prepared microcapsules, for example, using the technique koatservatsii or polymerization on the phase boundary, for example hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal systems of drug delivery to the site of action (for example, liposomes, albumen microspheres, microemulsions, nanoparticles and nanocapsules), or in microemulsion. Such methods are shown in Remington''s Pharmaceutical Sciences, supra.

Compositions which are intended for use in vivo must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or after lyophilization and the creation of the current song.

Therapeutic compositions described in this invention, is usually placed in a vial with sterile inlet, such as a container for intravenous solution or a vial having a stopper capable of with a needle for subcutaneous injection.

The route of administration is in accordance with known methods, such as intravenous, intraperitoneal injectively examples of compositions of continuous allocation include semi-permeable polymer matrices in the form of a definite form, for example, films, or microcapsules. Matrix of continuous allocation include polyesters, hydrogels, polylactide (U. S. Patent 3773919, EP 58481), copolymers of L-glutamic acid and ethyl-L-glutamate (U. Sidman et al. , Biopolymers 22(1), 547-556 (1983)), poly(2 - hydroxyethylmethacrylate) (R. Langer et al. , J. Biomed. Mater. Res. 15, 167-277 (1981) and R. Langer Chem. Tech. 12, 98-105 (1982)), ethylene vinyl acetate (R. Langer et al. id. ), or poly-D-(-)-3 - hydroxybutiric acid (EP A). Composition of continuous allocation also include liposomes. Liposomes containing molecules within the scope of the present invention, a receiving method, known from applications and patents: DE A; Epstein et al. , Proc. Nail. Acad. Sci. USA 82, 3688-3692 (1985); Hwang et al. , Proc. Nail. Acad Sci. USA 71, 4030-4034 (1980), EP A; EP A; EP A; EP A; EP A; Japanese patent application 83-118008; U. S. Patents 4485045 and 4544545; and EP A. Typically, liposomes are small (about 200-800 angstroms) single-disk types, in which the lipid content is more than 30 mol. % cholesterol, and the amount is adjusted for optimal therapy.

An effective amount of a molecule that is used therapeutically, depends, for example, from therapeutic objectives, the route of administration and the condition of the patient. Thus, for therapist needs to determine the dose and modify with whom I from about 1 μg/kg up to 100 mg/kg or more, depending on the above factors. Typically, the Clinician must enter the molecules described in this invention, until, until you reach a dose that will provide the desired biological effect. Achieve this therapy is easily monitored by conventional methods of analysis. If the goal of treatment is to reduce weight, it is usually the treatment lasts up until not achieved the desired body weight.

Not related to therapy the use of hybrid OB-protein antibodies described in this invention include their use for the identification and purification of OB receptor. Identification and expression cloning OB-receptor using OB-protein-immunoadhesin described in the examples below.

The invention will be further illustrated by the following, not bordering it, with examples.

Example 1. The expression of OB-immunoadhesins

Using the methods of genetic engineering, OB protein man was expressional as a fusion with the hinge, CH2 and CH3 regions of IgG-1. DNA constructs encoding the Chimera OB-protein of human rights and IgG-1 Fc-region, was created with clones Fc-region of IgG-1 person. OB cDNA person were obtained using polymerase reaction synthesis chain (PCR) from fat cells dtsdec man (product of Clontech Buick - Clone cDNA). IP is Noah length) OB-protein (amino acids 1-167, Fig. 5) and the sequence of the IgG-1 person, starting from aspartic acid 216, considering the amino acid at position 114 as the first residue of the constant region of the heavy chain (Kabat et al. , Sequences of Proteins of Immunoloqical Interest 4th ed. (1987)), which is the first residue of the hinge region of IgG-1 after the cysteine residue is included in the binding of heavy - light chain and ends with a residue at position 441 to enable CH2 and CH3 Fc region of IgG-1. There was a box of three codons of amino acids (GlyValThr) between OB-and IgG-1-coding sequences. If necessary, this short linker sequence can easily erase (delete), for example, using siteprovides deletion mutagenesis, to create an accurate binding (interface) between the coding sequences OB-protein and the hinge region of the IgG-1. The coding sequence of OB-IgG-1-immunoadhesin was subcloned into the vector pRK5tk-neo on the basis of pRK5, which contains a neomycin-breeding marker for temporal expression in 293 cells using the calcium phosphate method (Suva et al. , Science 237, 893-896 (1987)). 293 cells were cultured in HAM's: low glucose DMEM (modified by way of Dulbecco Wednesday Needle) medium (50: 50) containing 10% FBS and 2 mm L-Gln. To clean the S24 and the medium was collected after three days. Culture medium was filtered.

The filtered supernatant of 293 cells (400 ml) containing recombinant IgG OB-1 person, prepared 1 mm in phenylmethyl-sulfonyl fluoride containing 2 mg/ml Aprotinin. This substance was placed at 4oC 1 x 4.5 cm column with protein And immobilized on agarose (Pierce catalog N 20365); balanced 100 mm HEPES pH8.

The flow rate was 75 ml/h As only placed the sample, the column was washed balanced buffer up until AND280did not reach the baseline. OB-IgG-1 protein was suirable solution of 3.5 M MgCl2+ 2% glycerol (nezaloginennym) with a flow rate of 15 ml/h Eluate was collected with periodic stirring in 10 ml of 100 mm HEPES pH8, to reduce the concentration of MgCl2about 2 times and increase pH. Suirvey protein is then dialyzed against phosphate buffered saline, concentrated, sterilized and stored or when 4oC or frozen at -70oC. OB-IgG-1-immunoadhesin obtained by this method was determined by SDS-PAGE and its purity was above 90%.

Example 2. Research animals

A. materials and methods

The production of OB-protein - OB cDNA related to mice or rats, the warped sequence Zhang et al. , supra. Mature OB protein (amino acids 22-167) expressed in E. coli by inserting OB-coding sequence into the reading frame with secretory sequence of the E. coli thermostable enterotoxin II, in 5'-3' direction of the promoter of E. coli alkaline phosphatase (Chang et al. , Gene 55, 189-96 (1987)). After cell lysis, the insoluble fraction was dissolved in 8 M buffer with urea pH 8,35, in the presence of 25 mm DTT (dithiothreitol).

Restored OB protein was purified using gel filtration and liquid chromatography high resolution (HPLC) with reversed phase, then re-folded in the presence of glutathione. Re-folded OB protein was purified HPLC with reversed phase and analyzed using SDS-PAGE and amino acid and mass spectrometric analyses.

Receiving PEG-hOB - PEG-derivatives OB-protein man received reactions hOB, purified using chromatography with reversed phase, Succinimidyl derivative of PEG propionic acid (SPA-PEG), having a nominal molecular weight of 10 kD, which was obtained from Shearwater Polymers, Inc. (HuntsVille, AL). After cleaning the hOB protein using chromatography with reversed phase, approximately 1-2 mg/ml protein in 0.1% trifi to 8.5 with NaOH. SPA-PEG was added to the reaction mixture, bringing the molar ratio of protein to SPA-PEG to 1: 1 and 1: 2 and the mixture is incubated at room temperature for one hour. After the reaction and purification by gel-electrophoresis or ion exchange chromatography samples extensively dialyzed against phosphate buffered saline and sterilized by filtering through to 0.22 micron filter. The samples were stored at 4oC. Under these conditions, PEG-hOB obtained by the reaction at a molar ratio of protein to SPA-PEG equal to 1: 1, consisted mainly of molecules with one attached 10 D PEG with a small amount of 2 PEG - containing varieties. PEG-hOB at a molar ratio of 1: 2 consisted of approximately equal amounts of 2 and 3 PEG-s attached to the hOB as shown SDS gel electrophoresis. In both reactions also detected a small amount of unreacted protein. This unreacted protein, if necessary, can be easily removed using gel filtration or ion exchange. PEG derivatives OB-protein a person can also obtain essentially, after applying the aldehyde, the method described in European patent (EP 372752, published June 13, 1990).

Research animals. All mA what about the age C57BI/6J-ob/ob (ob/ob) female mice, genetically obese, bought in Jackson Labs (Bar Harbor, ME). Thin female mice of the same genetic background (SW/6) were purchased in Harlan Sprague Dawley (Hollister, CA). Mice were housed in groups of 3-6 with ad libitum access to water and standard food for mice (Purina 5010: Purina Mills, Richmond, in) family Boxing with controlled temperature, humidity and lighting (light from 06: 00 to 18: 00 hours).

Minimations pumps (Alzet model 2002: Alza Corp. , Palo Alto, CA) filled with purified recombinant (chimeric) OB protein (100 µg/kg/day) in sterile phosphate buffered saline (PBS) or PBS under sterile conditions after the instructions of the manufacturer, and incubated (kept) during the night in sterile saline at room temperature prior to implantation in mice. Mice were anestesiologi ketamine/xylazine, and minimations pumps injected drug subcutaneously in the mid-scapular region. Daily subcutaneous injection of purified recombinant (chimeric) OB-protein, hOB-IgG-1 hybrid protein or PBS was injected into the mid-scapular region healthy mice. Injection was carried out for one hour without lighting. Recorded the body weight of each mouse (accurate to 0.1 g) and the weight of food, soderjashie represented as means SEM. The number of animals as described below and in the inscriptions on the figures.

B. Results with continuous subcutaneous infusion of OB-protein

Thin female mice were treated OB protein related to mice or rats, or as a continuous subcutaneous infusion or daily subcutaneous injection. The results are shown in Fig. 1. The upper part of Fig. 1 (figs. 1A) shows that the OB protein is much more effective for weight loss when it comes to introduction in the form of a continuous infusion than when the same dose is given as a daily subcutaneous injection. The lower part of Fig. 1 (figs. 1B) shows the same difference in the ability of OB protein to reduce the weight of adipose tissue.

C. Results with IgG OB-1-Chimera

Obese female ob/ob mice were treated OB protein human or OB-IgG-1 Chimera relating to the human race. The data shown in Fig. 2. The data presented in the upper part of Fig. 2 (Fig. 2A), showed that hOB-IgG-1-hybrid protein is more effective than native hOB protein, to reduce body weight, when both proteins are introduced equally with daily subcutaneous injections. It should be noted that the increase in efficiency will be even more pronounced if the data is present in the confirm previous observation, that continuous subcutaneous infusion (pump) hOB-protein is more effective than daily subcutaneous injection (inj) to reduce body weight.

The data shown in the lower part of Fig. 2 (Fig. 2B) show that hOB-IgG-1-hybrid protein significantly reduces the consumption of food. This result was unexpected, because it was assumed that a hybrid protein is too large to cross the blood brain barrier and have an impact.

Obese (ob/ob) female mice were treated hOB or hOB-IgG-1-Chimera daily subcutaneous injection for 7 days. The data shown in Fig. 3, again showing that the Chimera is more effective than native hOB protein for weight reduction of the body (upper part of Fig. 3, Fig. 3A) and food intake (lower part of Fig. 3, Fig. 3B).

In an additional experiment, obese (ob/ob) female mice were treated or hOB-IgG-1 hybrid protein, native hOB or hCD4-IgG-1 (control) by daily subcutaneous injection for seven days. The results shown in Fig. 5, confirm that hOB-IgG-1-hybrid protein is more effective than native hOB protein for weight reduction of the body (upper and middle part of Fig. 5, Fig. 5A and Fig. 5B) and food consumption (Negotino person or PEG derivative OB, related to the human race. The data shown in Fig. 4. The data shown in the upper diagram of Fig. 4A, show that PEG-hOB more effective than native hOB protein to reduce body weight, when both protein injected similarly with the help of daily subcutaneous injections.

The data shown in the bottom diagram, Fig. 4B show that PEG-hOB protein is much more effective to reduce food intake than unmodified native hOB.

The control example. Identification and cloning of the OB-receptor

OB protein-immunoadhesin of example 1 was used for the discovery and expression of clone OB receptor. First, to determine the source of the receptor, organized the screening of cell lines with 1 mg/ml IgG OB-1 hybrid protein using flow cytometry. System identification, which consists of a biotinylated secondary antibody, followed by streptomycinresistant provides effective signal amplification and provides for the detection of cells expressing a small number of receptors. Found that two cell lines: 293 embryonic kidney cells human and A549 lung cells human contact OB IgG-1, but not with Flt-4 control bacterial expressing OB-protein man. The addition of 10 μg/ml OB man completely blocks the binding of IgG OB-1 from 293 cells.

To isolate cDNA coding for OB receptor, COSN cells was temporarily transfectional pools of approximately 105clones oligo dT premirovany 293 cells cDNA library in pRK5B. Transfection cells were enriched using panning on plates coated with Fc antibody against human, after incubation with IgG OB-1. After three cycles of enrichment is one of the thirty pools caused OB IgG-1-mediated adhesion COSN cells with connecting plates, which may compete for leptin person. cDNA clones randomly selected from the third cycle was transfectional in pools of 10-20. Individual clones was finally identified after the destruction of one pool of 10, what exactly were calculated using panning.

The sequence analysis revealed the clone from about 5300 bp with an open reading frame that encodes a protein of 896 amino acids. The sequence corresponded to the transmembrane protein type I signal peptide with a length of 22 amino acids of the extracellular region of 819 amino acids, a transmembrane region with 21 amino acids and a short intracellular region of 34 amino acids. Found taglia et al. , supra, and this sequence is identical to the sequence of the receptor of the person presented in the related application (Serial No. 08/585005 filed January 11, 1996).

Although the invention is illustrated using examples, the scope of the invention is not restricted by them. It is clear that other possible modifications and variations within the limits of the invention. It is assumed that all such modifications are within the scope of this invention.

All links, including the examples, therefore, are specifically included in the description of this invention.

The sequence listing is provided at the end of the description.

1. Covalent derivative of S-protein has a longer half-life in plasma and/or slower clearance than native S-protein (besity protein - protein responsible for obesity) and can reduce the body weight and/or food consumption at being treated individual.

2. Derived under item 1, characterized in that it is derived native S-protein of man.

3. Derived under item 1, characterized in that it is S-immunoglobulin Chimera.

4. Derived under item 1, characterized in that one is 2">

5. Derived under item 4, wherein the non-protein polymer is polyethylene glycol.

6. Composition for treatment of a condition associated with abnormal expression or function of the Ob gene, or for the detection of biological reactions (sensitivity) - mediated S-receptor, comprising an effective amount of S-derivative, under item 1.

7. The composition according to p. 6, effective for reducing weight and/or appetite.

8. The composition according to p. 6, effective to reduce elevated levels of insulin.

9. The method of treatment of a condition associated with abnormal expression or function of the Ob gene, or detecting a biological response (susceptibility), mediated S-receptor, including the introduction of the individual being treated, derived under item 1.

10. The method according to p. 9, wherein the condition being treated, which are selected from the group consisting of obesity, bulimia and diabetes I or type II.

11. The way to promote weight loss or loss of appetite in a subject, comprising the introduction of this subject an effective amount of a derivative, under item 1.

12. Chimeric polypeptide that includes the amino acid sequence of S-protein, str>13. Chimeric polypeptide under item 12, wherein the immunoglobulin sequence is a constant region.

14. Chimeric polypeptide under item 13, wherein the S-protein is human.

15. Chimeric polypeptide under item 14, characterized in that the merging of two RH-polypeptide - IgG - heavy chain is carried out using at least one disulfide bond with obtaining homodimeric immunoglobulinovogo patterns.

16. The chimeric polypeptide according to p. 15, characterized in that at least one of the merged S-polypeptide - IgG - heavy chain is linked to a light chain immunoglobulin.

17. The selected nucleic acid, the nucleotide sequence can encode a fusion S-protein - immunoglobulin.

18. Expressing the vector is capable of replication containing nucleic acid under item 17.

19. A host cell, for example, to obtain the chimeric polypeptide according to p. 12, transformable capable of replication expressing vector under item 18.

20. The method of production (obtain hybrid) protein responsible for obesity, immunoglobulin, comprising the following stages: a) transformation of a cell-is this merger, C) culturing the transformed host cells under item 19, so that they expressed this nucleic acid, and (C) the allocation of the merger.

21. The method according to p. 20, characterized in that the cells are the owners of cotransferred with nucleic acid that encodes at least two merge S-protein-immunoglobulin.

22. The method according to p. 21, characterized in that the cells are additionally transformed with a nucleic acid that encodes at least one light chain immunoglobulin.

23. The method of treatment of a condition associated with abnormal expression or function of the Ob gene, or detecting a biological response (susceptibility), mediated S-receptor comprising the administration to a patient a therapeutically effective amount of a chimeric polypeptide under item 12.

24. The method according to p. 23, wherein the condition is selected from the group consisting of obesity, bulimia and diabetes I or type II.

25. The composition for treating obesity, comprising an effective amount of a chimeric polypeptide under item 12 and a pharmaceutically acceptable carrier.

26. Way to stimulate the growth of cells expressing S-receptor, including the provision of interaction data CL 9,11 and 25.

 

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