WO2004028555A1 - Resistin binding proteins, their preparation and use - Google Patents

Abstract

The present invention relates to the use of Resistin Binding Proteins in the manufacture of medicaments, to certain such proteins and their preparation.

Description

Resistin Binding Proteins, their Preparation and Use

Field of the Invention The present invention relates to the use of resistin binding proteins in the manufacture of medicaments, to certain such proteins and their preparation.

Background of the Invention

Non-insulin-dependent diabetes mellitus (NIDDM) is a common, chronic disease that is a major cause of morbidity and mortality in industrialized societies. NIDDM affects 95% of diabetics and afflicts more than 5% of the world's population. NIDDM has a strong genetic component and is tightly-linked to obesity. The disorder is characterized by severe tissue resistance to the effects of insulin. Although impaired insulin secretion contributes to NIDDM, insulin levels are often increased in the early course of the disease. This peripheral insulin resistance is a major difference between NIDDM and insulin-dependent diabetes (Olefsky 1985, Olefsky et al. 1985). The resistance occurs despite qualitatively and quantitatively normal insulin receptors, thus implicating one or more defective steps in the insulin signaling pathway downstream from insulin binding to its receptor.

Nevertheless, although NIDDM is characterized by insulin resistance, the only available pharmacological treatments for NIDDM until only recently were insulin or agents that increase insulin secretion. New pharmacological approaches to treating NIDDM have been developed that target other metabolic abnormalities (Larkins 1997). For instance, the thiazolidinediones (TZDs) are a new class of orally active drugs that decrease insulin resistance by enhancing the actions of insulin at a level distal to the insulin receptor (Henry 1997). TZDs, which include troglitazone, pioglitazone, and rosaglitazone, are thought to sensitize target tissues to the action of insulin. These compounds are ineffective at lowering serum glucose levels in the absence of insulin. In animal models of NIDDM, TZDs lower plasma glucose levels, and decrease insulin . and triglycerides to near normal levels (Fujita et al 1983 and Fujiwara et al. 1988).

TZDs are generally well tolerated by patients although troglitazone therapy has been associated with hepatic dysfunction, which has been fatal in a few cases ( atkins et al. 1998). Thus, liver function tests should be monitored frequently during treatment, although the causal relationship and mechanism of TZD-mediated liver toxicity have not been established. Hence, there is still a need to find and develop other anti-diabetic compounds that will be safe and efficient.

Gestational Diabetes Mellitus (GDM) is diagnosed during pregnancy. In most cases, it disappears after birth. About two to five percent of pregnant women are diagnosed with GDM- about 180,000 women per year in the United States. It develops during the second trimester (about 24 to 26 weeks of pregnancy) and is manifested by insulin resistance. The mother's high blood sugar stimulates the fetus to make more insulin, absorb more glucose and gain extra weight. If unregulated, these changes can have serious and harmful effects on both mother and child.

Polycystic ovary syndrome (PCOS) is characterized by anovulation (irregular or absent menstrual periods) and hyperandrogenism (elevated serum testosterone and androstenedione4 Other changes associated with this condition include enlarged ovaries with an increased number of small (6- 10mm) follicles around the periphery (Polycystic Appearing Ovaries or PAO). PCOS is estimated to affect 6-10% of women. One of the major biochemical features of polycystic ovary syndrome is insulin resistance accompanied by compensatory hyperinsulinemia. There is increasing data that hyperinsulinemia produces the hyperandrogenism of polycystic ovary syndrome by increasing ovarian androgen production, particularly testosterone and androstenedione and by decreasing the serum sex hormone binding globulin concentration. The high levels of androgenic hormones interfere with the pituitary ovarian axis, leading to increased LH levels, anovulation, amenorrhea, and infertility. Hyperinsulinemia has also been associated high blood pressure and increased clot formation and appears to be a major risk factor for the development of heart disease, stroke and type II diabetes.

Recently, a protein named resistin was identified, isolated and cloned (WOOO/64920). According to this publication, resistin is secreted by adipocytes and administration of recombinant resistin to mice impairs glucose tolerance and insulin action. Importantly, TZDs inhibit resistin expression, suggesting at least one of their therapeutic mechanisms in diabetes. According to WOOO/64920, resistin is at least one of the factors that cause insulin resistance in obesity. Furthermore, antibodies to resistin enhanced insulin action in vitro, as measured by glucose uptake by adipocytes. However, therapeutic use of such antibodies to resistin may be problematic because such antibodies may act as antigens to elicit secondary immune responses.

Cytokine binding proteins (soluble cytokine receptors) correspond to the extracellular ligand binding domains of their respective cell surface cytokine receptors. They are derived either by alternative splicing of a pre-mRNA, common to the cell surface receptor, or by proteolytic cleavage of the cell surface receptor. Such soluble receptors have been described in the past, including among others, the soluble receptors of IL-6 and IFN-gamma (Novick et al. 1989), TNF (Engelmann et al. 1989 and 1990), IL-1 and IL-4 (Maliszewski et al. 1990), IFN-alpha/beta (Novick et al. 1994 and 1992) and others. One cytokine-binding protein, named osteoprotegerin (OPG, also known as osteoclast inhibitory factor - OCIF), a member of the TNFR/Fas family, appears to be the first example of a soluble receptor that exists only as a secreted protein (Anderson et al. 1997, Simonet et al 1997, Yasuda et al. 1998). Interleukin-18-binding protein (IL-18BP) (Novick et al. 1999) represents a second example of such a soluble receptor which exists only as a secreted protein. Stomatin is a 31 kDa integral membrane protein found in red blood cells and other cell types. Human stomatin consists of 3 domains; a short N-terminal domain of 24 amino acid residues, a membrane-associated domain of 29 amino acid residues and a cytoplasmic C-terminal domain of 234 amino acid residues. The N-terminal domain is probably also cytoplasmic, as it is phosphorylated by the cytoplasmic protein kinase A (Salzer et al.1993). Yet, in one review article the N-terminal peptide is considered to be extra-cellular (Stewart et al. 1997). However, pro- stomatin lacks a signal peptide and therefore the chance of its N-terminal domain to exit the cytoplasmic membrane are rather slim. Cysteine residue No. 29 within the membrane-associated domain is S-palmytoylated (Snyers et al. 1999). Genetic disorders in which this protein is missing result in hemolytic anemia called stomatocytosis. (Stewart et al. 1992). Yet, Stomatin-deficient mice do not have hemolytic anemia and stomatocytosis, hence stomatin deficiency and stomatocytosis result from a different genetic disorder (Zhu et al. 1999). Stomatin is thought to function as a negative regulator of univalent cation permeability. The molecular mechanism for stomatin function is unknown, but its cytoplasmic portion has been suggested to act as a ball and chain tether that can directly plug ion channels and may also interact with the cytoskeleton (Stewart et al.1993). Structurally, stomatin belongs to a super-family of proteins, which also include prohibitins and plant disease response genes that controls cell proliferation, ion channel regulation and apoptosis (Nadimpalli et al. 2000). Stomatin is closely associated with Glutl in the plasma membrane and over-expression of stomatin results in a depression in the basal rate of glucose transport (Zhang et al. 1999).

Summary of the Invention

The invention relates to the use of stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction (e.g. PEG-conjugated), fragment and salt thereof, capable of at least one of the following:

(i) binding to resistin, (ii) modulating the activity of resistin, or

(iii) blocking the activity of resistin, in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

In one aspect, the invention relates to the use of an expression vector comprising the coding sequence of stomatin, a mutein, variant,' fusion protein, circularly permutated derivative, or fragment thereof, in the manufacture of a medicament for the treatment and/or prevention of disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

In another aspect, the invention relates to the use of a vector for inducing and/or enhancing the endogenous production of stomatin in the manufacture of a medicament for the treatment and/or prevention of disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

In addition the invention relates to the use of a cell that has been genetically modified to overexpress stomatin, a mutein, variant, fusion protein, circularly permutated derivative, or fragment thereof, in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

The invention provides pharmaceutical compositions for treating or preventing a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking a mutein, variant, fusion protein, circularly permutated derivative, active fraction (e.g. PEG-conjugated), fragment and salt thereof and mixtures thereof capable of at least one of the following: (i) binding to resistin, (ii) modulating the activity of resistin, or (iii) blocking the activity of resistin.

The invention also provides pharmaceutical compositions comprising an antibody (polyclonal antibody, monoclonal antibody, anti-idiotypic antibody, humanized antibody or a fragment, isoform, mutein, or functional derivative thereof ) that immunospecifϊcally binds to stomatin a mutein, variant, fusion protein, circularly permutated derivative, active fraction (e.g. PEG- conjugated), or fragment thereof, and a pharmaceutically acceptable carrier for treating or preventing disease which require modulation or blocking of resistin activity.

The invention also relates to pharmaceutical compositions comprising an expression vector encoding the sequence of stomatin a mutein, variant, fusion protein, circularly permutated derivative or fragment thereof and salt thereof, in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity. In one aspect, the invention provides a pharmaceutical composition comprising a vector including regulatory sequences functional in the cells for inducing and/or enhancing the endogenous production of stomatin in a cell in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

In another aspect, the invention provides a pharmaceutical composition comprising a cell that has been genetically modified to overexpress stomatin a mutein, variant, fusion protein, circularly permutated derivative, or fragment thereof, in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

In addition, the invention provides methods for treating or preventing a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity, comprising administering to a subject in which such treatment or prevention is desired, the above compositions.

In one embodiment, an antibody (polyclonal antibody, monoclonal antibody, anti-idiotypic antibody, chimeric antibody, humanized antibody or its fragments, isoforms, muteins, or functional derivatives) prepared using the protein of the invention is provided.

In another embodiment, a process for the purification of resistin is provided comprising: passing a sample trough a chromatographic column to which stomatin a mutein, variant, fusion protein, circularly permutated derivative, active fraction (e.g. PEG-conjugated) or fragment thereof is coupled, washing the column to remove unbound protein in the sample, and recovering the bound resistin. Description of the Figures

Figure 1 shows an autoradiogram of SDS-PAGE (10 % acrylamide; reducing conditions) of

12₅ complexes consisting of I-resistin (apparent molecular weight 10 kD), cross-linked to itself or to different cells. Following cross-linking to cells, the cells were washed, solubilized and immuno-precipitated with polyclonal anti resistin anti serum: Lane 1 : Cross-linking of

125

I-resistin to murine 3T3 F442 cells grown in medium containing fetal bovine serum. Lane 2:

125

Cross-linking of I-resistin to muπne 3T3 F442 cells grown in medium containing bovme serum. Lanes 3 and 7: molecular mass markers (indicated on the right sides of the gels in kD).

125 , 125 Lanes 4 and 8: cross-linking of I-resistin to itself. Lane 5: Cross-linking of I-resistm to

125 human HeLa cells. Lane 6: Cross linking of I-resistin to human foreskin fibroblasts. The specific cross-linked complexes of both human and mouse cells had a molecular mass of about 40 kD (indicated by arrows on the left sides of the gels).

125 Figure 2 shows a saturation-binding curve of I-resistin to its receptor on murine 3T3 F442 fibroblasts.

Figure 3 shows silver-stained SDS-PAGE (10% acrylamide; reducing conditions) of affinity purified fraction (Elution2) containing enriched cell-associated resistin binding proteins (ResBP) from HeLa cells. Lane 1 is the Elution 2 fraction. The full arrow indicates specific resistin binding proteins of molecular mass 28-32 kD. Lane 2 shows the molecular mass markers, indicated on the right side (in kD).

Figure 4 shows an autoradiogram of SDS-PAGE (10 % acrylamide; reducing conditions) of

125

I resistin (apparent molecular weight 10 kD), cross-linked to the following preparations and immunoprecipitated by anti resistin antibodies coupled to Protein-G: Lane 1 : Crude HeLa cell extract (Load fraction of the resistin affinity column) Lane 2: Unbound fraction (Wash fraction). Lane 3: Elution 2 fraction of the resistin affinity column (see Example 1). Lane 4: Elution 2 fraction, cross-linked in the presence of an excess of unlabeled resistin. The specific cross-linked products (of about 40 and 32 kD) in lane 3 are indicated by arrows and molecular weight markers are indicated on the right side (in kD).

Detailed Description of the Invention

The present invention relates to the use of stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or a fragment thereof and salt thereof for the treatment or prevention of insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases or conditions which require modulation or blocking of resistin activity or in cases of complications due to exogenously administered resistin. The term "stomatin"includes a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or fragment thereof and salt thereof

The present invention also relates to pharmaceutical compositions prepared for administration of stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or fragment thereof and salt thereof by mixing stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or fragment thereof and salt thereof, with physiologically acceptable carriers, and/or stabilizers and/or excipients, and prepared in dosage form, e.g., by lyophilization in dosage vials.

The present invention further relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof in any disease in which endogenous production or exogenous administration of resistin induces the disease or aggravates the situation of the patient.

The pharmaceutical compositions may comprise a pharmaceutically acceptable carrier and e.g., a viral vector such as any one of said AAN-based viral vectors or another vector expressing stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or fragment thereof and suitable for administration to humans and other mammals for the purpose of attaining expression in vivo of stomatin a mutein, variant, fusion protein, circularly permutated derivative, or fragment thereof f, i.e. for use in gene therapy.

Accordingly, stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof or vectors expressing same in vivo are indicated for the treatment of insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases or conditions which require modulation or blocking of resistin activity or in cases of complications due to exogenously administered resistin.

The method of administration can be via any of the accepted modes of administration for similar agents and will depend on the condition to be treated, e.g., intravenously, intramuscularly, and subcutaneously, by local injection or topical application, or continuously by infusion, etc. The amount of active compound to be admimstered will depend on the route of administration, the disease to be treated and the condition of the patient. Local injection, for instance, will require a lower amount of the peptide on a body weight basis than will intravenous infusion.

Stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof and vectors for expressing such stomatin , in humans and other mammals may be employed in the treatment, alleviation or prevention of conditions in which resistin is involved or caused by an excess of exogenously administered or endogenously produced resistin. Such conditions are, e.g., insulin resistance, gestational diabetes, polycystic ovary syndrome and NIDDM.

As used herein, the expression "modulating the activity of resistin" means the capability of stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof to modulate any resistin activity other than blocking, e.g. partial inhibition, enhancement, or the like.

As used herein, the expression "blocking the activity of resistin" refers to blocking any biological activity of resistin. The biological activity of stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof is measured by adding it at different concentrations to a biological assay for resistin. For example, inhibition of insulin- induced adipocyte differentiation by resistin is one possible biological assay for resistin. The resistin blocking activity of stomatin can be exemplified by the ability of stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof to block the resistin associated inhibition of insulin-induced increase of glucose uptake by rat adipocytes.

The terms "treating" and "preventing", as used herein, should be understood as partially or totally preventing, inhibiting, attenuating, ameliorating or reversing one or more symptoms or cause(s) of the disease. When "treating" with the substances according to the invention are given after the onset of the disease, "prevention" relates to administration of the substances before signs of disease can be noted in the patient.

In addition to the use stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof, it can, of course, also be employed for the purification of resistin itself. For this purpose, stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof is coupled to an affinity column and crude resistin is passed through. The resistin can then be recovered from the column by, e.g., elution at low pH.

Isolation and enrichment of stomatin was carried out in accordance with the invention by ligand- affinity chromatography. Accordingly, isolation stomatin, was carried out by passing a detergent extract of human cells, e.g., human HeLa cells through a chromatographic column to which resistin was coupled, washing the column to remove unbound components of the sample, and eluting the bound stomatin.

According to the present invention a human resistin having an additional lysine residue at its N- terminus, produced in E. coli was coupled to agarose and packed into a column. Human HeLa cells (10¹⁰) were solubilized by a lysis buffer containing detergents. The clarified cell lysate was loaded onto the column, the column was washed thoroughly and bound proteins were then eluted at pH 2.2 in the presence of a detergent and immediately neutralized. SDS-PAGE (10%) under reducing conditions and silver staining gave bands in the range of 28-32 kD. The resistin binding protein obtained in the column eluate was identified as a resistin binding protein by its ability to specifically cross-link with 1 ^^I-resistin. Resistin binding protein was further characterized by mass spectrometry (MS). For this purpose, the eluted proteins in Fraction 2 of the resistin affinity column was subjected to preparative SDS-PAGE followed by silver staining. Stained protein bands in the range of 28-32 kD were excised, the protein was electroeluted from the gel, subjected to digestion with trypsin and the resulting peptide mixture was analyzed by liquid chromatography (LC) coupled to tandem MS-MS. By this method one cell associated resistin binding protein was identified and found to be identical with a previously cloned protein termed human stomatin, Genebank Accession No. P27105 (http://www.ncbi.nlm.nih.gov).

The affinity-purified Resistin binding protein retained the ability to bind its labeled ligand (4 5ι__resj_sti_n)₅ and following covalent cross-linking, a complex of resistin (10 kD) and Resistin binding protein (about 30 kD) of molecular mass of about 40 kD was apparent by SDS-PAGE and autoradiography.

To test for a domain within stomatin which is capable of binding resistin, peptides of human stomatin can be prepared, by solid-phase peptide synthesis or by recombinant technology and may be screened to block the biological activity of resistin in a bioassay. Thus, a specific stomatin peptide can be found that may inhibit the biological activity of resistin when added to culture of pre-adipocytes i.e. it enhances their insulin-induced differentiation into adipocyte-like cells even in the presence of resistin.

Studies of cross-linking of ¹²⁵I-resistin to mouse and human cells revealed a specific cross-linked complex of apparent molecular mass of about 40 kD comprising resistin (10 kD) and a cell associated protein ( of about 30 kD). The cell associated ResBP was shown to have high affinity to resistin (about Kd of 10^"10M). This cell associated resistin binding protein could be stomatin.

As used herein, the expression "binding to resistin" means the capability of stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof to bind resistin, e.g. as evidenced by its binding to labeled resistin when affinity-purified e..g. as in Example 6 herein.

Stomatin fragments synthesis can be synthetically produced. However, fusion proteins containing stomatin-fragments are preferably prepared by recombinant DNA methods. For recombinant expression, the DNA encoding stomatin fragment, optionally with an appropriated signal peptide sequence can be inserted in an expression vector, preferably in a vector in which the stomatin- fragment will be produced as a fusion protein with a larger polypeptide, e.g., immunoglobulin G Fc domain and optionally a specific tag such as Histidine x6, to facilitate its purification.

Thus, the invention relates to stomatin fragment with an affinity of ~10^"10M to resistin, or its muteins or their fragments, optionally fused to another polypeptide, and to functional derivatives or active fractions thereof.

The present invention also encompasses variants of said proteins of the invention. Preferred variants are the ones having at least 80% amino acid sequence identity, a more preferred variant is one having at least 90% identity and a most preferred variant is one having at least 95% identity to the amino acid sequence of said proteins of the invention. The term "sequence identity" as used herein means that the amino acid sequences are compared by alignment according to Hanks and Quinn (1991) with a refinement of low homo logy regions using the Clustal-X program, which is the Windows interface for the ClustalW multiple sequence alignment program (Thompson et al., 1994). The Clustal-X program is available over the internet at ftp://ftp-igbmc.u-strasbg.fr/pub/clustalx/. Of course, it should be understood that if this link becomes inactive, those of ordinary skill in the art could find versions of this program at other links using standard internet search techniques without undue experimentation. Unless otherwise specified, the most recent version of any program referred herein, as of the effective filing date of the present application, is the one, which is used in order to practice the present invention. Another method for determining "sequence identity" is he following. The sequences are aligned using Nersion 9 of the Genetic Computing Group's GDAP (global alignment program), using the default (BLOSUM62) matrix (values -4 to +11) with a gap open penalty of -12 (for the first null of a gap) and a gap extension penalty of -4 (per each additional consecutive null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the claimed sequence.

Muteins in accordance with the present invention include those encoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA under stringent conditions and which encode said protein in accordance with the present invention, comprising essentially all of the naturally- occurring sequences encoding for example stomatin and fragments thereof comprising regions responsible for binding resistin.

The term "hybridization" as used herein shall include any process by which a strand of nucleic acid joins with complementary strand through a base pairing (Coombs J, 1994, Dictionary of Biotechnology, stokton Press, New York NY). "Amplification" is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction technologies well known in the art (Dieffenbach and Dveksler, 1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY). "Stringency" typically occurs in a range from about Tm-5°C (5°C below the melting temperature of the probe) to about 20°C to 25 °C below Tm.

The term "stringent conditions" refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as "stringent". See Ausubel et al., Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, NY, 1987-1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

As used herein, stringency conditions are a function of the temperature used in the hybridization experiment, the molarity of the monovalent cations and the percentage of formamide in the hybridization solution. To determine the degree of stringency involved with any given set of conditions, one first uses the equation of Meinkoth et al. (1984) for determining the stability of hybrids of 100% identity expressed as melting temperature Tm of the DNA-DNA hybrid: Tm = 81.5 C + 16.6 (LogM) + 0.41 (%GC) - 0.61 (% form) - 500/L where M is the molarity of monovalent cations, %GC is the percentage of G and C nucleo tides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. For each 1 C that the Tm is reduced from that calculated for a 100%) identity hybrid, the amount of mismatch permitted is increased by about 1%. Thus, if the Tm used for any given hybridization experiment at the specified salt and formamide concentrations is 10 C below the Tm calculated for a 100%) hybrid according to the equation of Meinkoth, hybridization will occur even if there is up to about 10%o mismatch. As used herein, "highly stringent conditions" are those which provide a Tm which is not more than 10 C below the Tm that would exist for a perfect duplex with the target sequence, either as calculated by the above formula or as actually measured. "Moderately stringent conditions" are those, which provide a Tm, which is not more than 20 C below the Tm that would exist for a perfect duplex with the target sequence, either as calculated by the above formula or as actually measured. Without limitation, examples of highly stringent (5-10 C below the calculated or measured Tm of the hybrid) and moderately stringent (15-20 C below the calculated or measured Tm of the hybrid) conditions use a wash solution of 2 X SSC (standard saline citrate) and 0.5%> SDS (sodium dodecyl sulfate) at the appropriate temperature below the calculated Tm of the hybrid. The ultimate stringency of the conditions is primarily due to the washing conditions, particularly if the hybridization conditions used are those, which allow less stable hybrids to form along with stable hybrids. The wash conditions at higher stringency then remove the less stable hybrids. A common hybridization condition that can be used with the highly stringent to moderately stringent wash conditions described above is hybridization in a solution of 6 X SSC (or 6 X SSPE (standard saline-phosphate-EDTA), 5 X Denhardt's reagent, 0.5% SDS, 100 µ g/ml denatured, fragmented salmon sperm DNA at a temperature approximately 20 to 25 C below the Tm. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC (Ausubel, 1987, 1999).

As used herein the term "muteins" refers to analogs of stomatin or fragment thereof in which one or more of the amino acid residues of a natural stomatin are replaced by different amino acid residues, or are deleted, or one or more amino acid residues are added to the natural sequence of stomatin or fragment thereof, without changing considerably the capability of the resulting products as compared with the wild type stomatin to bind to resistin. These muteins are prepared by known synthesis and/or by site-directed mutagenesis techniques, or any other known technique suitable thereof.

Any such mutein preferably has a sequence of amino acids sufficiently duplicative of that of a stomatin such as to have substantially similar activity to stomatin. One activity of stomatin is its capability of binding resistin. As long as the mutein has substantial binding activity to resistin, it can be used in the purification of resistin, such as by means of affinity chromatography, and thus can be considered to have substantially similar activity to stomatin. Thus, it can be determined whether any given mutein has substantially the same activity as stomatin by means of routine experimentation comprising subjecting such a mutein, e.g., to a simple sandwich competition assay to determine whether or not it binds to an appropriately labeled resistin, such as radioimmunoassay or ELISA assay or by cross linking to radiolabelled resistin as exemplified below.

In a preferred embodiment, any such mutein has at least 40% identity or similarity with the amino acid sequence of stomatin. More preferably, it has at least 40%, at least 60%>, at least 70%), at least 80%> or, most preferably, at least 90%> identity or similarity thereto. Muteins of stomatin polypeptides or muteins of which can be used in accordance with the present invention, or nucleic acid coding therefore, include a finite set of substantially corresponding sequences as substitution peptides or polynucleotides which can be routinely obtained by one of ordinary skill in the art, without undue experimentation, based on the teachings and guidance presented herein. For a detailed description of protein chemistry and structure, see Schulz, G.E. et al., Principles of Protein Structure, Springer-Nerlag, New York, 1978; and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are hereby incorporated by reference. For a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al, supra, at §§ A.1.1 -A.1.24, and Sambrook et al, supra, at Appendices C and D. Preferred changes for muteins in accordance with the present invention are what are known as "conservative" substitutions. Conservative amino acid substitutions of stomatin polypeptides may include synonymous amino acids within a group which have sufficiently similar physicochemical properties that substitution between members of the group will preserve the biological function of the molecule, Grantham, Science, Vol. 185, pp. 862-864 (1974). It is clear that insertions and deletions of amino acids may also be made in the above-defined sequences without altering their function, particularly if the insertions or deletions only involve a few amino acids, e.g., under thirty, and preferably under ten, and do not remove or displace amino acids which are critical to a functional conformation, e.g., cysteine residues, Anfinsen, "Principles That Govern The Folding of Protein Chains", Science, Vol. 181, pp. 223-230 (1973). Peptides and muteins produced by such deletions and/or insertions come within the purview of the present invention. Preferably, the synonymous amino acid groups are those defined in Table I. More preferably, the synonymous amino acid groups are those defined in Table II; and most preferably the synonymous amino acid groups are those defined in Table -H.

TABLE I

Preferred Groups of Synonymous Amino Acids Amino Acid Synonymous Group

Ser Ser, Thr, Gly, Asn

Arg Arg, Gin, Lys, Glu, His

Leu He, Phe, Tyr, Met, Nal, Leu

Pro Gly, Ala, Thr, Pro Thr Pro, Ser, Ala, Gly, His, Gin, Tin- Ala Gly, Thr, Pro, Ala Nal Met, Tyr, Phe, He, Leu, Nal Gly Ala, Thr, Pro, Ser, Gly He Met, Tyr, Phe, Nal, Leu, He Phe Trp, Met, Tyr, He, Nal, Leu, Phe

Tyr Trp, Met, Phe, He, Nal, Leu, Tyr

Cys Ser, Thr, Cys

His Glu, Lys, Gin, Thr, Arg, His

Gin Glu, Lys, Asn, His, Thr, Arg, Gin Asn Gin, Asp, Ser, Asn

Lys Glu, Gin, His, Arg, Lys

Asp Glu, Asn, Asp

Glu Asp, Lys, Asn, Gin, His, Arg, Glu

Met Phe, He, Nal, Leu, Met Trp Trp

TABLE H

More Preferred Groups of Synonymous Amino Acids Amino Acid Synonymous Group

Ser Ser

Arg His, Lys, Arg

Leu Leu, He, Phe, Met

Pro Ala, Pro Thr Thr

Ala Pro, Ala

Nal Nal, Met, He

Gly Gly

He He, Met, Phe, Nal, Leu Phe Met, Tyr, He, Leu, Phe

Tyr Phe, Tyr

Cys Cys, Ser

His His, Gin, Arg

Gin Glu, Gin, His Asn Asp, Asn

Lys Lys, Arg

Asp Asp, Asn

Glu Glu, Gin

Met Met, Phe, He, Nal, Leu Trp Trp TABLE HI

Most Preferred Groups of Synonymous Amino Acids Amino Acid Synonymous Group

Ser Ser

Arg Arg

Leu Leu, He, Met

Pro Pro

Thr Thr

Ala Ala

Nal Nal

Gly Gly

He He, Met, Leu

Phe Phe

Tyr Tyr

Cys Cys, Ser

His His

Gin Gin

Asn Asn

Lys Lys

Asp Asp

Glu Glu

Met Met, He, Leu

Trp Trp

Examples of production of amino acid substitutions in proteins which can be used for obtaining muteins of stomatin polypeptides, or muteins for use in the present invention include any known method steps, such as presented in US patents RE 33,653, 4,959,314, 4,588,585 and 4,737,462, to Mark et al; 5,116,943 to Koths et al., 4,965,195 to Νamen et al; 4,879,111 to Chong et al; and 5,017,691 to Lee et al; and lysine substituted proteins presented in US patent No. 4,904,584 (Shaw et al).

In another preferred embodiment of the present invention, any mutein of stomatin has an amino acid sequence essentially corresponding to that of a stomatin. The term "essentially corresponding to" is intended to comprehend peptides with minor changes to the sequence of the natural peptide which do not affect the basic characteristics of the natural peptides, particularly insofar as their ability to bind resistin. The type of changes which are generally considered to fall within the "essentially corresponding to" language are those which would result from conventional peptide synthesis, resulting in a few minor modifications, and screening for the desired activity in the manner discussed above. In addition to binding resistin, the muteins may also modulate and/or block resistin activity.

The term "circularly permuted" as used herein refers to a linear molecule in which the termini have been joined together, either directly or through a linker, to produce a circular molecule, and then the circular molecule is opened at another location to produce a new linear molecule with termini different from the termini in the original molecule. Circular permutations include those molecules whose structure is equivalent to a molecule that has been circularized and then opened. Thus, a circularly permuted molecule may be synthesized de novo as a linear molecule and never go through a circularization and opening step. The particular circular permutation of a molecule is designated by brackets containing the amino acid residues between which the peptide bond is eliminated. Circularly permuted molecules, which may include DNA, RNA and protein, are single-chain molecules, which have their normal termini fused, often with a linker, and contain new termini at another position. See Goldenberg, et al. I. Mol. Biol., 165: 407- 413 (1983) and Pan et al. Gene 125: 111-114 (1993), both incorporated by reference herein. Circular permutation is functionally equivalent to taking a straight-chain molecule, fusing the ends to form a circular molecule, and then cutting the circular molecule at a different location to form a new straight chain molecule with different termini. Circular permutation thus has the effect of essentially preserving the sequence and identity of the amino acids of a protein while generating new termini at different locations.

The term "fragment" refers to fragments of stomatin capable of binding resistin or capable of modulating its activity, e.g. an extracellular domain of a cell associated stomatin, obtainable by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques. The term "fusion protein" refers to a polypeptide comprising stomatin or a mutein or fragment thereof, fused with another protein, which, e.g., has an extended residence time in body fluids, stomatin may thus be fused to another protein, polypeptide or the like, e.g., an immunoglobulin or a fragment thereof. It may also be fused to a high molecular weight polymer, such as polyethylene glycol (PEG) in order to prolong residence time.

The term "salts" herein refers to both salts of carboxyl groups and to acid addition salts of amino groups of resistin , a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or a fragment thereof . Salts of a carboxyl group may be formed by means known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid. Of course, any such salts must have substantially similar activity to stomatin.

"Functional derivatives" as used herein cover derivatives of stomatin and its muteins and fusion proteins and fragment thereof, which may be prepared e.g. from the functional groups which occur as side chains on the residues, or by adding groups to the N- or C-terminus, by any means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the peptide which is substantially similar to the activity of stomatin and do not confer toxic properties on compositions containing it.

The above derivatives may, for example, include polyethylene glycol side-chains (PEG- conjugated), which may mask antigenic sites and extend the residence of a stomatin peptide in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of free amino groups of the amino acid residues formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example that of seryl or threonyl residues) formed with acyl moieties. As "fragment" of stomatin , muteins and fusion proteins, the present invention covers any fragment or precursors of the polypeptide chain of the peptide molecule alone or together with associated molecules or residues linked thereto, e.g., sugar or phosphate residues, or aggregates of the peptide molecule or the sugar residues by themselves, provided said active, fraction substantially retains the capability of binding resistin. Various recombinant cells such as prokaryotic cells, e.g., E. coli, or eukaryotic cells, such as CHO, yeast or insect cells can produce fusion proteins containing stomatin-N or its derivatives. Methods for constructing appropriate vectors, carrying DNA that codes for stomatin such as stomatin or a mutein, fused protein, or fragment thereof e.g. stomatin-N or its muteins, fragments or fusion proteins thereof and suitable for transforming (e.g., E. coli, mammalian cells and yeast cells), or infecting insect cells in order to produce a recombinant stomatin a mutein, fused protein, or fragment thereof are well known in the art. See, for example, Ausubel et al., eds. "Current Protocols in Molecular Biology" Current Protocols. 1993; and Sambrook et al, eds. "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor Press, 1989.

The invention also relates to a cell that has been genetically nodified to produce recombinant stomatin a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or a fragment thereof and salt thereof.

For the purpose of expressing fusion proteins containing stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or a fragment thereof their DNA and the operably linked transcriptional and translational regulatory signals, are inserted into vectors which are capable of integrating the desired gene sequences into the host cell chromosome. In order to be able to select the cells, which have stably integrated the introduced DNA into their chromosomes, one or more markers which allow for selection of host cells which contain the expression vector is used. The marker may provide for prototrophy to an auxotropic host, biocide resistance, e.g., antibiotics, or resistance to heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by cotransfection. Additional elements may also be needed for optimal synthesis of single chain binding peptide mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

Said DNA molecule to be introduced into the cells of choice will preferably be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Such plasmids and vectors are well known in the art (Bollon, D. P., et al. 1980 J. Clin. Hematol. Oncol. 10:39-48, Botstein, D., et al. 1982 Miami Wint. Symp. 19:265-274, Broach, J. R., in "The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance", Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 445-470 (1981), Broach, J. R., 1982. Cell 28:203-204. and Maniatis, T., in "Cell Biology: A Comprehensive Treatise, Vol. 3: Gene Expression", Academic Press, NY, pp. 563-608 (1980).). Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the expression vector may be introduced into an appropriate host cell by any of a variety of suitable means, such as transformation, transfection, lipofection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, etc.

Host cells to be used in this invention may be either prokaryotic or eukaryotic. Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas,

Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537),

E. coli W3110 (F^", lambda", phototropic (ATCC 27325). Under such conditions, the protein will not be glycosylated. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.

Preferred eukaryotic hosts are mammalian cells, e.g., human, monkey, mouse and Chinese hamster ovary (CHO) cells, because they provide post-translational modifications to protein molecules including correct folding, correct disulfide bond formation, as well as glycosylation at correct sites. Also yeast cells and insect cells can carry out post-translational peptide modifications including high mannose glycosylation.

A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids, which can be utilized for production of the desired fusion proteins in yeast and in insect cells. Yeast and insect cells recognize leader sequences on cloned mammalian gene products and secrete mature stomatin or a mutein, fused protein, or fragment thereof. After the introduction of the vector, the host cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of stomatin, or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment thereof. The expressed proteins are then isolated and purified by any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like, or by affinity chromatography, using, e.g., an anti- stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof, monoclonal antibodies immobilized on a gel matrix contained within a column. Crude preparations containing said recombinant stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof are passed through the column whereby stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof will be bound to the column by the specific antibody, while the impurities will pass through. After washing, the protein is eluted from the gel under conditions usually employed for this purpose, i.e. at a high or a low pH, e.g. pH 11 or pH 2.

The invention further relates to vectors useful for expression of stomatin or a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof in mammals and more specifically in humans e.g. for gene therapy. Vectors for short and long-term expression of genes in mammals are well known in the literature. Studies have shown that gene delivery to e.g., skeletal muscle, vascular smooth muscle and liver result in systemic levels of therapeutic peptides. Skeletal muscle is a useful target because of its large mass, vascularity and accessibility. However, other targets and particularly bone marrow precursors of immune cells have been used successfully. Currently available vectors for expression of fusion proteins in e.g., muscle include plasmid DNA, liposomes, protein-DNA conjugates and vectors based on adenovirus, adeno-associated virus and herpes virus. Of these, vectors based on adeno- associated virus (AAV) have been most successful with respect to duration and levels of gene expression and with respect to safety considerations (Kessler, P.D. 1996, Proc. Natl. Acad. Sci. USA 93, 14082-14087).

Procedures for construction of an AAN-based vector have been described in detail (Snyder et al, 1996, Current Protocols in Human Genetics, Chapters 12.1.1-12.1.17, John Wiley & Sons) and are incorporated into this patent. Briefly plasmid psub201, containing the wild-type AAN genome is cut with the restriction enzyme Xba I and ligated with a construct consisting of an efficient eukaryotic promoter, e.g., the cytomegalovirus promoter, a Kozak consensus sequence, a DΝA sequence coding for stomatin or its muteins or fusion proteins or fragments thereof, a suitable 3' untranslated region and a polyadenylation signal, e.g., the polyadenylation signal of simian virus 40. The resulting recombinant plasmid is cotransfected with a helper AAN plasmid e.g., pAAN/Ad into mammalian cells e.g., human T293 cells. The cultures are then infected with adenovirus as a helper virus and culture supernatants are collected after 48-60 hours. The supernatants are fractionated by ammonium sulfate precipitation, purified on a CsCl density gradient, dialyzed and then heated at 56°C to destroy any adenovirus, whereas the resulting recombinant AAV, capable of expressing stomatin or its muteins or fusion proteins remains stable at this step. The use of a vector for inducing and/or enhancing the endogenous production of stomatin, in a cell normally silent for expression of stomatin, or expressing amounts of stomatin which are not sufficient, are also contemplated according to the invention. The vector may comprise regulatory sequences functional in the cells desired to express the stomatin. Such regulatory sequences comprise promoters or enhancers. The regulatory sequence is then introduced into the right locus of the genome by homologous recombination, thus operably linking the regulatory sequence with the gene, the expression of which is required to be induced or enhanced. The technology is usually referred to as "endogenous gene activation" (EGA), and it is described e.g. in WO 91/09955.

The invention also contemplates the use of a pharmaceutical composition comprising a cell that has been genetically modified to overexpress stomatin or a mutein, variant, fusion protein, circularly permutated derivative, and fragment thereof an isoform, a mutein, fused protein, or functional derivative thereof, in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

So far, the physiological role of the soluble cytokine receptors has not been established. The soluble receptors bind their specific ligands and in most cases inhibit their biological activity, as is shown, e.g., in the TNF system (7, 8). In very few cases, e.g., IL-6, the soluble receptor enhances the biological activity. The recombinant soluble TNF receptor, also known as TBP (TNF binding protein) is found to prevent septic shock in animal models, while soluble forms of IL-1 receptor are found to have profound inhibitory effects on the development of in vivo alloreactivity in mouse allograft recipients. Similarly, a soluble fragment of stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or fragment and salt thereof, may find use as modulators of resistin activity, e.g. in insulin resistance, gestational diabetes, polycystic ovary syndrome, and NDDDM.- They may thus be used, e.g. in any disease in which endogenous production or exogenous administration of resistin induces the disease or aggravates the situation of the patient. The invention also includes antibodies against stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or fragment thereof and salt thereof and their use for the treatment or prevention of insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases or conditions which require modulation or blocking of resistin activity or in cases of complications due to exogenously administered resistin. The term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (MAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, and humanized antibodies as well as fragments thereof provided by any known technique, such as, but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques. A specific ELISA may be developed using a pair of antibodies obtained from different species e.g. rabbit and mouse antibodies.

The invention will now be illustrated by the following non-limiting examples:

Examples

Example 1 : Preparation of a radio-iodinated human resistin derivative.

A mammalian expression vector for human resistin, to which the tag KYWSHPQFEK (SEQ ID: NO 2) (comprising the Strep-tag H, which is a streptavidin binding sequence) is added at the C- terminus, was prepared. The resulting resistin derivative was expressed in HEK293 cells and purified on a Streptavidin-agarose column. It gave a single band of 10 Kd as determined by SDS- PAGE (12%) acrylamide) under reducing conditions. This preparation was found to be biologically active (see below) and was used for radioiodination by the following modification of the Chloramine T method. The whole procedure was performed at 4°C. Resistin (20 μg in 50 μl PBS containing 20%> glycerol) was mixed with an equal volume of 0.5 M phosphate buffer pH 7.5 [A]. Chloramine T solution (50 μl, 1 mg/ml in H₂O) was mixed with 1 mCi of [¹²⁵I] Nal in 10 μl for 20 sec [B]. Mix [A] was added to Mix [B] and incubated for 20 sec. The reaction was stopped by the addition of NaHSO₃ (5 mg/ml, 50 μl) and KI (5 mg/ml, 50 μl) for 2 min. Free iodine was separated from the radio-iodinated resistin by a size exclusion column (Sephadex G- 25, PD-10 column, Amersham-Pharmacia) pre-equilibrated with 25 ml PBS containing 0.25% gelatine and 0.02% NaN₃. The eluted fractions (1 ml each) were monitored by a gamma-counter counter. Peak fractions had a specific activity of 3X10¹⁷ cpm/mol.

Example 2: Identification of a cell-associated resistin binding protein by cross-linking to radioiodinated resistin and immunoprecipitation.

Cross-linking studies of human 425]_resistin were carried out with mouse cells. Murine 3T3 F442 fibroblasts (Green and Kehindle 1975 Cell 5 19-27 and Cell 7 105-113) can be differentiated into mature adipocytes following treatment with insulin. Since fetal bovine serum (FBS), unlike bovine serum (BS), contains insulin, a change in medium supplementation to the cells from BS to FBS will trigger differentiation into adipocytes.

Murine 3T3 F442 fibroblasts (0.5-2x10⁸ cells) grown in either FBS or BS were washed with PBS and then incubated with 425τ__resj_stιn (12x10^ cpm) for 1.5 hr at 4°C. DSS (final concentration of 2 mM) was added for 20 min. The cells were then washed with excess PBS and solubilized for 1 hr at 4°C by a lysis buffer (1% Triton X 100 in 10 mM CHAPS, 20 mM Tris HC1, pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM Benzamidine, 1 mM PMSF, 6 μg/ml aprotinin). The lysate was spun (15,000xg, 4°C for 20 min) and the clarified supernatant was immunoprecipitated with anti human resistin rabbit antibodies (Example 8) bound to Protein-G agarose beads overnight at 4°C. The beads were then washed with 0.1% Triton X 100 in PBS and boiled with SDS-PAGE-Sample Buffer containing 25 mM DTT. The supernatant was analyzed by SDS-PAGE (10% acrylamide) followed by autoradiography. A band of molecular weight of about 40 kD, probably consisting of approximately a 30 kD cell surface protein cross-linked to the 10 kD ^-^1-τesist , was observed (Fig. 1).

In similar manner, Human HeLa cells (4x10^) and human foreskin fibroblasts (cells 6x10^) were washed with PBS, cross-linked with human 4 5j__resj_sti_n (33χi()6 cpm), washed, solubilized, immunoprecipitated and analyzed by SDS-PAGE to investigate whether a cell-associated ResBP exists. A band of molecular weight of about 40 kD, probably consisting of approximately a 30 kD cell surface receptor cross-linked to the 10 kD 125j__resi_sti_{nj was} observed (Fig. 1). The cross-linking studies carried with whole cells show that a cell surface ResBP of apparent molecular weight of about 30 kD is present in both human and mouse cells.

Example 3: Determination of the affinity of humanl25ι__resj_s i_n to its cell-associated binding sites.

Murine 3T3 F422 cells (5X10⁴) in DMEM containing 10%o bovine serum (BS) were seeded in a

24 well plates. On the next day, the cells were washed with DMEM containing 2% BS and 0.1%) sodium azide (diluent) and incubated with the diluent for 20 minutes at room temperature.

Medium was discarded and a series of dilutions of 1 ^^I-resistin (3xl0¹⁷ dpm/mol) in the diluent (12x10 -3x10 cpm, 0.5 ml) were added to the wells and incubated for 90 min at room temperature with occasional shaking. Wells were quickly washed twice with the diluent and once with PBS, cells were collected by trypsin and the radioactivity was counted by a gamma counter. The binding affinity was determined by Scatchard analysis using the Ligand program. A dissociation constant (Kd) of 10^"10M was obtained. The saturation binding curve is shown in Fig. 2. The results show that cell associated ResBP has high affinity to resistin (of about Kd of 10^"10M).

Example 4: Isolation of a membrane-associated resistin binding protein.

A human resistin having an additional lysine residue at its N-terminus, produced in E. coli was obtained from Peprotech Inc. Rocky Hills NJ and was found to be biologically active by its ability to inhibit the differentiation of murine 3T3-F442 cells into adipocytes. This lysine- modified resistin (2 mg) was coupled to Affigel-10 (1 ml, BioRad), according to the manufacturer's instructions and packed into a column. Human HeLa cells (10¹⁰) grown in DMEM containing 10% bovine serum were washed with phosphate buffered saline (PBS) and solubilized for 1 hr at 4°C by a Lysis Buffer (1% Triton X 100, 10 mM CHAPS, 20 mM Tris HC1, pH 7.5, 150 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM Benzamidine, 1 mM PMSF, 6 μg/ml aprotinin). The cell lysate was spun at 14,000xg for 30 min at 4°C and the supernatant was loaded onto the column at a flow rate of 0.25 ml/min. The column was washed with 250 ml 0.5M NaCl in PBS containing 0.1% Triton X-100. Bound proteins were then eluted with 25 mM citric acid, pH 2.2 containing 0.1% Triton X-100 and benzamidine (1 mM). Fractions of 1 ml were collected and immediately neutralized by 1M Na2CO3. The fractions were analyzed by SDS-PAGE (10%)) under reducing conditions and the protein bands were visualized by silver (Fig. 3) staining. Bands in the range of 28~32kD (in line with the 30 kD protein found in the cross linked product) were excised and subjected to mass spectrometric analysis.

Example 5: Molecular Characterization of a cell-associated resistin binding protein by Mass Spectrometry.

The specific protein band obtained in Example 4 (in the range of 28-32 kD) were excised from the gel, the proteins were electroeluted and digested with trypsin. The resulting tryptic digest was subjected to liquid-chromatography and tandem mass spectrometry (LC-MS/MS). One tryptic peptides provided unequivocal sequences: VQNATLAVANITNΆDSATR (SEQ ID NO:3). A computerized search in the NCBI protein database using this sequence with the Blast program has identified resistin binding protein to be the 30 kD human stomatin (SEQ ID NO:l) Genebank Accession No. P27105 (http://www.ncbi.nlm.nih.gov). Additional proteins corresponding to human beta actin, tropomyosin 4, voltage-dependent anion channel 1, tropomodulin 3 and immunoglobulin kappa and beta chains were also identified in the same protein band.

Example 6: Cross-linking of affinity-purified resistin-BP to radioiodinated resistin.

Samples (50 μl) from the ligand affinity purification step were incubated (6 hours at 4°C) with 425τ__resi_sri_n (1,800,000 cpm). Disuccinimidyl suberate (DSS), dissolved in dimethyl sulfoxide (Me2SO, 20 mM), was then added to a final concentration of 2 mM and the mixture was left for 20 min. at 4°C. The reaction was stopped by the addition of 1 M Tris-HCl pH 7.5, and 1 M NaCl to a final concentration of 100 mM. Rabbit anti resisitin antibodies (50 μl) coupled to Protein-G sepharose beads (50 μl) and washed with PBS containing 0.1%ι Triton X-100 were added and the mixture was incubated overnight at 4°C with a gentle rotation. The beads were then washed 4x with PBS containing 0.1% Triton X-100. A sample buffer containing Dithiothreitol (DTT, 25 mM final concentration) was added and the mixtures were analyzed by SDS-PAGE (10% acrylamide) followed by autoradiography (Fig. 4). A specific band having an apparent molecular mass of about 40 kD was obtained in fractions eluted from the resistin affinity column (lanes 3) but not in the column wash (lane 2), containing all other crude cellular proteins. This band probably consisted of approximately a 30 kD resistin binding protein cross-linked to the 10 kD 125j__resi_stin. χ_ne ba^ _was attenuated if an excess of unlabeled resistin was added to the cross-linking reaction (lane 4). An additional band of 32 kD was observed in the elution fraction and was partially abolished by the addition of excess unlabeled resistin. This band probably consisted of approximately a 22 kD resistin binding protein cross-linked to the 10 kD 125j__resi_smι# Alternatively these bands are cross linked complexes comprising a dimer of 425j__resj_sri_n (20 kD) and two resistin binding proteins of 20 kD and 12 kD.

Example 7: measuring the biological activity of resistin and stomatin.

The biological activity of resistin (Peprotech) was measured by its ability to inhibit the differentiation of murine pre-adipocytes (3T3 F422) into adipocytes induced by switching the supplementation of culture growth medium from bovine serum (BS) to fetal bovine serum (FBS).

Addition of ResBP to the resisitin may be used to study its effect on resistin activity.

For the bioassay, murine 3T3 F442 cells were grown in 24 well plates (5xl0⁴/well) in DMEM containing 10% BS. When cells reached confluency the BS was replaced with 10% FBS. At day 8 differentiation into adipocytes was observed by the appearance of oil droplets in the cytoplasm.

Cells were observed microscopically for 7 more days and then stained with Oil Red, which highlights the oil droplets. Resistin was assayed for its ability to inhibit this differentiation. Upon switching of the cultures to medium containing FBS, a resisitin stock (1 μg/ml) in DMEM containing 10% FBS was serially diluted down to 7.5 pg/ml (in two-fold dilutions) and the dilutions were added to the cells. It was found that resistin at a concentration of 70 ng/ml completely inhibited the differentiation of 3T3 F442 cells into adipocytes.

The biological activity of stomatin is measured by adding it at different concentrations together with resistin and checking its effect on the biological activity of resistin. Example 8: Preparation of polyclonal antibodies to resistin.

Rabbits were injected subcutaneously with 12 μg of a pure resistin preparation, emulsified in complete Freund's adjuvant. Three weeks later, they were injected again subcutaneously with 12 μg of the resistin preparation in incomplete Freund's adjuvant. Three additional injections of resistin as solution in PBS were given at 10-day intervals. The rabbits were bled 10 days after the last immunization. The development of resistin specific antibody was followed by a radioimmunoassay. ^^l-labeled resistin (100,000 cpm) was mixed with various dilutions (1:200, 1:1000, 1 :5,000 1:25,000 and 1:125,000) of the rabbit serum. A suspension of protein-G agarose beads (20 μl, Pharmacia) was added in a total volume of 200 μl and agitated for 1 and a half hour at room temperature. The beads were then washed 3 times and bound radioactivity was counted. Preimmune serum was used as a negative control. The titer of the resistin antiserum was >1:125,000.

Example 9: Preparation of monoclonal antibodies to stomatin.

Female Balb/C mice (3 months old) are first injected with 2 μg purified stomatin in an emulsion of complete Freund's adjuvant, and three weeks later, subcutaneously in incomplete Freund's adjuvant. Three additional injections of 2μg each are given at 10-day intervals, subcutaneously in PBS. Final boosts of 2μg are given intraperitoneally, 4 and 3 days before the fusion, to the mouse showing the highest binding titer as determined by IRIA (see below). Fusion is performed using NSO/1 myeloma cell line and lymphocytes extracted from both the spleen and lymph nodes of the animal as fusion partners. The fused cells are distributed into microculture plates and the hybridomas are selected in DMEM supplemented with HAT and 15% horse serum. Hybridomas that are found to produce antibodies to stomatin are subcloned by the limiting dilution method and injected into Balb/C mice that had been primed with pristane for the production of ascites. The isotypes of the antibodies are defined with the use of a commercially available ELISA kit (Amersham, UK).

The screening of hybridomas producing anti-stomatin monoclonal antibodies is performed as follows: Hybridoma supernatants are tested for the presence of anti-stomatin antibodies by an inverted solid phase radioimmunoassay (IRIA). 96 well microtiter flexible plates (Dynatech Laboratories, Alexandria, VA) are coated with homogenous stomatin (10 μg/ml, 100 μl/well). Following overnight incubation at 4°C, the plates are washed twice with PBS containing BSA (0.5%) and Tween 20 (0.05%) and blocked in washing solution for at least 2 hrs at 37°C. Hybridoma culture supernatants (100 μl/well) are added and the plates are incubated for 4 hrs at 37°C. The plates are washed 3 times and a ^I25I-goat-anti-mouse (100,000 cpm/well) is added for 4 hrs at room temperature. The plates are washed 3 times, wells are cut and counted in a gamma counter.

Alternatively the hybridomas can be screened as follows: 96-well micro titer plates are coated with goat anti-mouse antibodies and blocked as above. Hybridoma supernatants are added and the plates incubated for 4 hrs at 37°C. The plates are washed 3 times and a ¹²⁵I-stomatin (100,000 cpm/well) is added for 4 hrs at room temperature. The plates are washed 3 times, wells are cut and counted in a gamma counter.

Example 10: Purification of stomatin by affinity chromatography with monoclonal antibodies.

Antibodies against stomatin are utilized for the purification of stomatin by affinity chromatography. Ascitic fluid containing the monoclonal antibody secreted by the hybridoma is purified by ammonium sulfate precipitation at 50%> saturation followed by extensive dialysis against PBS. About 10 mg of immunoglobulins are bound to 1 ml Affigel 10 (BioRad USA), as specified by the manufacturer.

250 ml of human proteins (equivalent to 250 1 of crude urine) are loaded on 0.5 ml of the anti stomatin antibody column at 4°C at a flow rate of 0.25 ml/min. The column is washed with PBS until no protein is detected in the washings, stomatin is eluted by 25 mM citric acid buffer, pH 2.2 (8 x 1 column volume fractions) and immediately neutralized by 1 M Na2CO3. Further purification of this preparation is obtained by size exclusion chromatography.

Example 11: ELISA test.

Microtiter plates (Dynatech or Maxisorb, by Nunc) are coated with anti-stomatin monoclonal antibody (serum free hybridoma supernatant or ascitic fluid immunoglobulins) overnight at 4°C. The plates are washed with PBS containing BSA (0.5%) and Tween 20 (0.05%) and blocked in the same solution for at least 2 hrs at 37°C. The tested samples are diluted in the blocking solution and added to the wells (100 μl/well) for 4 hrs at 37°C. The plates are then washed 3 times with PBS containing Tween 20 (0.05%) followed by the addition of rabbit anti-stomatin serum (1:1000, 100 μl/well) for further incubation overnight at 4°C. The plates are washed 3 times and a conjugate of goat-anti-rabbit horseradish peroxidase (HRP, Jackson Labs, 1:10,000, 100 μl/well) is added for 2 hrs at room temperature. The plates are washed 4 times and the color is developed by ABTS (2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid, Sigma) with H2O2 as a substrate. The plates are read by an automatic ELISA reader.

The foregoing description of the specific embodiments reveal the general nature of the invention so that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation.

Alternatively, a sandwich ELISA test is developed as above, but instead of the polyclonal antibody another monoclonal antibody is used. The monoclonal antibody used for detection is biotinylated and detected by HRP-streptavidin.

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Claims

CLAIMS:

1. The use of stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof, capable of at least one of the following: (i) binding to resistin, (ii) modulating the activity of resistin, or (iii) blocking the activity of resistin, in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

2. The use according to claim 1, wherein stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, or fragment thereof, is PEG-conjugated.

3. The use of an expression vector comprising the coding sequence of stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof, in the manufacture of a medicament for the treatment and/or prevention of disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

4. The use of a vector for inducing and/or enhancing the endogenous production of stomatin in the manufacture of a medicament for the treatment and/or prevention of disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

5. The use of a cell that has been genetically modified to overexpress stomatin, or a mutein, fused protein, or fragment thereof in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

6. A pharmaceutical composition for treating or preventing a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity comprising stomatin, a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof and mixtures thereof capable of at least one of the following: (i) binding to resistin, (ii) modulating the activity of resistin, or (iii) blocking the activity of resistin.

7. A pharmaceutical composition comprising an antibody that immunospecifically binds to stomatin or a mutein, fused protein, or fragment thereof and a pharmaceutically acceptable carrier for treating or preventing disease which require modulation or blocking of resistin activity.

8. A pharmaceutical composition according to claim 7, wherein the antibody is a polyclonal antibody, monoclonal antibody, anti-idiotypic antibody, humanized antibody or a fragment, isoform, mutein, or functional derivative thereof.

9. A pharmaceutical composition comprising an expression vector encoding the sequence of stomatin, a mutein, variant, fusion protein, circularly permutated derivative, or fragment thereof in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

10. A pharmaceutical composition comprising a vector including regulatory sequences functional in the cells for inducing and/or enhancing the endogenous production of stomatin in a cell, in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

11. A pharmaceutical composition comprising a cell that has been genetically modified to overexpress stomatin a mutein, variant, fusion protein, circularly permutated derivative, or fragment thereof, in the manufacture of a medicament for the treatment and/or prevention of a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity.

12. A method for treating or preventing a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NIDDM and other diseases which require modulation or blocking of resistin activity, comprising administering to a subject in which such treatment or prevention is desired, the composition according to anyone of claims 9 7tol2 in an amount sufficient to treat or prevent said disease in the subject.

13. A method for treating or preventing a disease selected from insulin resistance, gestational diabetes, polycystic ovary syndrome, NDDDM and other diseases which require modulation or blocking of resistin activity, comprising administering to a subject in which such treatment or prevention is desired, comprising administration of stomatin a mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof.

14. A stomatin mutein, variant, fusion protein, circularly permutated derivative, active fraction, fragment and salt thereof, capable of at least the following:

(i) binding to resistin, (ii) modulating the activity of resistin, or (iii) blocking the activity of resistin,

15. A process for the purification of resistin comprising:

Passing a sample trough a chromatographic column to which stomatin or a fragment, fusion protein or fragment thereof or functional derivative thereof is coupled, washing the column to remove unbound protein in the sample, and recovering the bound resistin.