WO2006125632A2 - Anticorps de type agonistes qui se lient au recepteur tweak fn14 et modulent par consequent les phenotypes associes a l'adiposite, et leur utilisation a des fins therapeutiques - Google Patents

Anticorps de type agonistes qui se lient au recepteur tweak fn14 et modulent par consequent les phenotypes associes a l'adiposite, et leur utilisation a des fins therapeutiques Download PDF

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WO2006125632A2
WO2006125632A2 PCT/EP2006/004974 EP2006004974W WO2006125632A2 WO 2006125632 A2 WO2006125632 A2 WO 2006125632A2 EP 2006004974 W EP2006004974 W EP 2006004974W WO 2006125632 A2 WO2006125632 A2 WO 2006125632A2
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adipocytes
activating
antibody
antibodies
receptor
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PCT/EP2006/004974
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WO2006125632A3 (fr
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Dieter Link
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Rechtsanwalt Dr. Martin Prager Als Insolvenzverwalter Über Das Vermögen Der Xantos Biomedicine Ag, Pluta Rechtsanwalts Gmbh
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Priority claimed from EP05011275A external-priority patent/EP1764109A1/fr
Application filed by Rechtsanwalt Dr. Martin Prager Als Insolvenzverwalter Über Das Vermögen Der Xantos Biomedicine Ag, Pluta Rechtsanwalts Gmbh filed Critical Rechtsanwalt Dr. Martin Prager Als Insolvenzverwalter Über Das Vermögen Der Xantos Biomedicine Ag, Pluta Rechtsanwalts Gmbh
Publication of WO2006125632A2 publication Critical patent/WO2006125632A2/fr
Publication of WO2006125632A3 publication Critical patent/WO2006125632A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen

Definitions

  • Agonistic antibodies that bind to the Tweak receptor Fn14 and thereby modulate adiposity-associated phenotypes as well as their use in therapy
  • the invention relates to pharmaceutical compositions as well as their use for activating the signal pathway of the receptor Fn 14 (synonyms are also TWEAK receptor, TWEAK- R or TNFRSF12A) for interfering and/or modulating the differentiation of adipocytes and for use in adiposity/obesity and/or metabolic syndrome.
  • the invention relates to antibodies that inhibit the differentiation of pre-adipocytes to mature adipocytes and/or the storage of lipid molecules into differentiating or mature adipocytes by receptor- mediated signal transduction.
  • the invention relates to defined epitopes of the receptor Fn14 that induce signal transduction of the receptor upon specific antibody binding.
  • the invention relates to antibodies that are directed against defined activating epitopes on the receptor Fn14 and that act as Fn14- activating agents. Preferably, this activation can take place alone or, also, in combination with other Fn 14- activating agents. Furthermore, the invention relates to pharmaceutical compositions and their use for activating the signal pathway of the receptor Fn14, for interfering and/or modulating the differentiation of adipocytes, in particular, for the prevention and/or treatment of adiposity/obesity.
  • adiposity (obesity) and type Il diabetes are two of the most important metabolic diseases that pose an increasingly serious problem, in particular, in the western world. Furthermore, tendencies for a pandemia have also been observed for type Il diabetes.
  • Adiposity is a condition that is characterized by the overly accumulation of fat tissue in the body.
  • the adipocytes i.e. those cells that form the fat tissue of the body, are essential for the formation of fat tissue, and, therefore, for developing an obese phenotype.
  • adiposity is a chronic health dysfunction that is based on a polygenetic predisposition and also caused by environmental factors and is accompanied by a high accompanying and follow-up morbidity.
  • adiposity is associated with a significant risk, in particular, the development of type Il diabetes. Therefore, a long term treatment and care concept for adiposity is indispensable.
  • BMI body mass index
  • the classification of the BMI is established by the WHO (World Health Organization) in dependency of the mortality rate.
  • the normal BMI (18.5 - 24.4 kg/ m 2 ) lies in the range with the lowest relative mortality risk.
  • the BMI values 25 and 30 kg/ m 2 are the corresponding risk- oriented limits for adults for establishing overweight or (clinical) adiposity.
  • Overweight and adiposity go along with follow up or accompanying diseases (comorbidity). They are a substantial risk factor for the development of type Il diabetes. Frequently, they also result in social isolation and discrimination. They can assist or cause psychic disturbances and, thereby, reduce life expectancy and life quality.
  • the mortality increases by 1/3, at 40 kg/ m 2 by a factor 3, at more than 40 kg/ m 2 by a factor 3 - 20 according the weight.
  • the risk of cancer is also increased (gynaecological carcinomas (endometrial carcinoma - 4 times; mamma- and cervix carcinoma - 2 times), prostate-, gall bladder-, colon- carcinoma)).
  • the BMI as an assessment standard for adiposity does not consider the composition of a body's tissue. An increased muscle mass, water content or bone mass can effect the assessment and may be considered optionally.
  • the body's composition of the compartments - fat and fat-free mass may be included.
  • body liquid is not considered here
  • the normal range is given for a fat mass ⁇ 20% and a fat-free body mass of > 80%.
  • An increased fat content is described as adiposity.
  • an increase in extracellular mass or extracellular water relative to the fat-free body mass indicates adiposity.
  • studies such as the bioelectric impedance analysis (BIA) or the infrared spectroscopy (NIRI) are suitable methods and methods used by practitioners for determining the body fat status.
  • the fat distribution is important for the risk assessment.
  • adiposity must be regarded as an essential risk factor for the development of type Il diabetes, the diagnosis and therapy of adiposity already in early childhood is the most important measure to avoid, for example, the development of paediatric Il diabetes.
  • the BMI can be used in analogy to adults for assessing overweight and adiposity. Because the BMI in childhood and adolescence is influenced in accordance to the physiological changes of the body's fat mass percentage by distinct age- and gender- specific characteristics, one should take into account the age and gender for ones assessment.
  • the BMI percentile can be used (obtained by extrapolation) that converges at the age of 18 to a BMI of 25 kg/ m 2 (overweight) or 30 kg/ m 2 (adiposity).
  • the 90th or the 97th age- and gender-specific percentile can be used as limit for defining overweight or adiposity.
  • the BMI values 25 and 30 kg/ m 2 are the corresponding risk- related limits for adults.
  • For a BMI of 25 kg/m 2 the relative risk for men for acquiring type Il diabetes is 2.2, for women, it is even 5.5.
  • a treatment of obesity and adiposity is targeted either at slowing down a further increase in weight, but it is preferred to prevent it completely, or, in addition, it is preferred to reduce the body weight.
  • the same is particularly true for the amount of fat tissue of the individual to be treated.
  • the opposite is true for the treatment of a cachexia or lipodystrophy.
  • a treatment of obesity or a tendency for adiposity is preferably indicated for individuals with a BMI of more than 25 in view of the already increased risk of co- morbidities due to overweight. This is particularly true for the case where co-morbidities have already manifested themselves: in particular, when the development of insulin resistance or type Il diabetes is indicated.
  • individuals having a BMI of more than 30 without or with co-morbidities that are characterized as having clinical adiposity are treated. This is especially preferred for individuals with a high level of adiposity.
  • adiposity can be prevented in individuals with a normal BMI or a BMI that qualifies them as overweight, in particular in the case, when there are reasons for assuming that these individuals will tend to develop an adiposity later (increase of the BMI to more than 30 kg/ m 2 ) with an increased probability, in particular, an adiposity associated with co-morbidities.
  • these reasons may based on a genetic pre-disposition, a sustained dysfunction of the lipid- or lipoprotein metabolism (dyslipidaemia), a life style that can hardly be changed and that assists the development of adiposity, and/or an adiposity due to other diseases.
  • a diagnostic system can be helpful that allows for detecting, for example, genetic pre-dispositions directly or indirectly or modified amounts or activities of the receptor Fn14 or its natural ligand or parameters depending thereon, for example, in blood or in fat tissue.
  • diabetes in particular type Il diabetes.
  • Diabetes mellitus (generally known as "sugar disease") is a chronic metabolic disease that is characterized by an increased level of sugar in the blood. Different causes of the disease and also different disease characteristics require the discrimination of two types, type I and type Il diabetes.
  • Type I diabetes (formerly: juvenile diabetes) most often begins in adolescence and develops because of an immunological destruction of islet cells of the pancreas. These islet cells produce the hormone insulin that is responsible for the utilization of glucose from food. From the destruction of the islet cells an absolute insulin deficiency results. The glucose in the food cannot be metabolized any more and the blood level of sugar increases.
  • the treatment of type I diabetes is done by the administration of insulin.
  • the type Il diabetes (formerly: adult or old age diabetes) regularly develops at a later age. It is characterized in that body cells, where the insulin is supposed to function, do not react sufficiently to insulin. Amongst others, this may be ascribed to a resistance of adipocytes and skeletal muscle cells for insulin. Interestingly, mostly the older more mature adipocytes are resistant, whereas younger adipocytes regularly do not have an insulin resistance.
  • Such an insulin resistance is considered to be the result of prolonged increased blood sugar and insulin levels as they are, for example, observed in overweight people.
  • the therapy of type Il diabetes is done stepwise: at first a diet for lowering the blood sugar level in general is attempted. If these dietary measures are not sufficient for treatment, subsequently medicaments lowering blood sugar are administered, and in advanced stages insulin is administered, too.
  • TNFSF12 protein TNFSF12
  • TWEAK protein TNFSF12
  • the modulation of the differentiation of adipocytes by an activation of the TNFSF12 signal transduction system is pursued as a therapeutic strategy independently of whether the TNFSF12/Fn14 signal transduction system is involved in the disposition, aetiology or associated symptoms of the disease.
  • Aberrant TNFSF12 expression or aberrant Fn14 receptor activity and/or signal transduction are no requirements or obstacles for performing the therapeutic method described herein.
  • TNFSF12 can be employed for the treatment of adiposity. It was demonstrated that TNFSF12 is capable of reversibly inhibiting the differentiation or the function of murine and also primary human adipocytes and to obtain a reduction of fat deposits in vivo. This allows for the first time the treatment of adiposity on the level of fat cell differentiation.
  • This protein TNFSF12 unfolds a therapeutic effect preferably by directly affecting certain fat tissues within an individual or also indirectly by other fat tissue compartments or also by the interaction with other tissues that are involved in the regulation of the energy homoeostasis.
  • TNFSF12 The explanation for the found phenotype of the protein TNFSF12 results from the property of TNFSF12 as a receptor ligand that can elicit signal transduction upon binding:
  • TNFSF12 is a cell surface-associated type Il membrane protein of 249 amino acids in length that was originally described as a member of the tumor necrosis factor (TNF) superfamily (Review in: Wiley et al., Cytokine & Growth Factor Reviews, 14, page: 241 - 249, 2003).
  • TNF tumor necrosis factor
  • a biologically active part is cleaved into the extracellular environment as a soluble protein with a length of about 156 amino acids and this can in turn activate the corresponding receptor.
  • This receptor activation itself can initiate changes in the cell, for example, assist cell division, enhance cellular migration and lead to the secretion of cytokines that are discussed in the context of inflammatory or cytotoxic processes.
  • DR3 was described as a receptor for TNFSF12 (Marsters et al., Cunr. Biol., 8, page: 525 - 528, 1998). Later it was demonstrated that DR3 is not the physiological receptor for TNFSF12 (Schneider et al., Europ. J. Immun., 29, page: 1785 - 1792, 1999; and Kaptein et al., FEBS Lett, 485, page:135 - 141 , 2000). Instead, there is only one receptor known for TNFSF12 in the present state of the art that is bound by TNFSF12 with physiological activity (Wiley et al., Immunity, 15(5), page: 837 - 846, 2001).
  • This receptor a member of the TNF superfamily, is described as ,,TWEAK- R" or ..fibroblast growth factor inducible 14" (Fn 14) (Meighan-Mantha et al., J. Biol. Chem., 274, page: 33166 - 33176, 1999; Wiley et al., Cytokine & Growth Factor Reviews, 14, page: 241 - 249, 2003).
  • This receptor is the smallest member of the group of the TNF-receptors. At present no other ligands are known that bind Fn14 (Wiley et al., Cytokine & Growth Factor Reviews, 14, page: 241 - 249, 2003).
  • TNFSF12 does not bind to other known TNF-receptors (Wiley et al., Cytokine & Growth Factor Reviews, 14, page: 241 - 249, 2003).
  • the primary sequence of Fn14 comprises 129 amino acids, wherein the amino terminus of the receptor is processed so that the mature protein consists for 102 amino acids only.
  • TNFSF12 TNFSF12
  • inflammatory cytokines such as, for example, IL- 6, IL-8 and RANTES has also described inter alia in fibroblasts (Chicheportiche, Arthritis Res, 4, page: 126 - 133, 2002).
  • TWEAK naturally passes the blood-brain barrier when it is expressed there in infiltrating leucocytes.
  • TWEAK-mRNA a negative regulation of TWEAK-mRNA was observed in vivo in lipopolysaccharide-induced inflammatory, acute and chronic reaction and animal models for autoimmune diseases (Chicheportiche et al., Biochem. Biophys. Res. Comm., 279, page: 162-165, 2000).
  • the transgenic animals either produced a first a cell-bound, full-length
  • TWEAK protein or a soluble TWEAK protein fragment.
  • TWEAK protein led to thrombosis in the heart chambers and to their dilatation, to hyperplasia of cells of the bile duct and oval cells in the liver as well as to an enhanced vacuole formation and cell death of liver cells.
  • cysts were formed in the glomeruli of the kidneys.
  • lung granulomatoses and the penetration of lymphocytes were recognized in the context of an inflammation.
  • WO 03/086311 pursued antagonistic therapeutic strategies that are aimed at the inhibition of the TWEAK-dependent signal transduction.
  • a genetic inflammatory disease in kidneys with signs of Alport syndrome that is inter alia characterized by the thinning and final destruction of the membranes of the glomeruli by fibrotic substitute tissue is provided.
  • an increased Fn 14 expression was shown in WO 03/086311 and a treatment of animals with said kidney changes with TWEAK antagonists (antagonistic antibodies) led to a reduction of the pathological fibrotic changes.
  • WO 03/086311 an increased expression of TWEAK protein in vivo seems to correlate with pathological diagnosis in the heart, the liver, the kidney and the lung, which in turn requires a blockage of TWEAK-dependent activities in the therapeutic application.
  • WO 03/086311 it was also described that in vitro the exposition of murine pre- adipocytes (3T3-L1 cells) to TNFSF12 can interfere negatively with their ability to incorporate lipids intracellular ⁇ . The mechanism is unclear because it was not shown in vivo whether Fn 14 is the only receptor for TNFSF12 and whether an expression of Fn 14 was present in 3T3-L1 cells or also in fat tissue was given. This is particularly relevant because Polek et al. (J. Biol. Chem., 278, page 32317 - 23, 2003) still discuss the possibility of several receptors for TWEAK.
  • murine pre-adipocytes typically display a so-called clonal expansion. This is an increase in the cell count due to a short lived re-entry into the cell cycle after induction for differentiation in previously post-mitotic cells. This phenomenon is not shown by differentiating human adipocytes. Factors that inhibit a clonal expansion can block the differentiation in murine adipocytes. It is therefore unclear whether factors inhibiting differentiation that were identified for murine adipocyte cell cultures can also achieve a blockage of differentiation in primary human adipocytes.
  • a dose-dependent and reversible inhibition of the differentiation of adipocytes or the inhibition of the lipid storage in human adipocytes was shown as a result of the TNFSF12-mediated Fn14 signal transduction next to the above described known effects of the TNFSF12/Fn14 ligand receptor system.
  • This inhibition of the differentiation of adipocytes was surprisingly shown by the applicant in fresh human fat cells (or fat precursor cells).
  • agonistic antibodies that bind to the receptor Fn 14 and activate it can elicit a direct effect on the differentiation of fat cells and inhibit fat cells differentiation.
  • TNF-R and other TNF-like receptors the activation of the Fn14 signal pathway preferably occurs when many receptors in close proximity are expressed on the cell surface. This process is known as receptor cluster formation.
  • the TNF ligands are multivalent complexes that can bind to more than one receptor simultaneously and aggregate in this way. Receptor cluster formation as a means for receptor activation has been elaborately described in other systems, in particular, for tyrosine kinase (Ullrich and Schlessinger, Cell, 61, page 203 - 212 (1990); Kolanus et al., Cell, 74, page 171 - 183 (1993)).
  • Fn14 ligands and/or Fn14-activating agents that also comprise, for example, antibodies, antibody derivatives, binding proteins or binding molecules that can induce the cluster formation and/or a downstream signalling on the surface of target cells can be useful for directly stimulating the Fn14 signal pathway in these cells.
  • the signal pathway for Fn14 is known amongst others for activating reaction pathways that can potentially modulate immunological or inflammatory of cytotoxic processes.
  • These properties of the Fn14 activation as well as the ability to inhibit these effects by antagonistic antibodies are the basis for the use of Fn14-blocking agents, including specific antagonistic Fn 14 antibodies for the treatment of diseases that are associated with immunological or inflammatory or cytotoxic processes as described by Biogen in WO 03/086311.
  • the use of agonistic antibodies is described in WO03/086311 that effect the induction of the Fn14 signal transduction. Whether such an activation is detrimental for normal tissue due to induced immunological or inflammatory or cytotoxic processes still remains unclear in the state of the art.
  • Ligands such as TN FSF 12 that activate the receptor Fn 14 can directly inhibit the differentiation of fat cells by ligand/receptor interactions and the subsequent receptor activation and signal transduction. Because receptor activation can also be achieved by agonistic antibodies, for example, those that recognize the corresponding receptor Fn 14 and induce signal transduction upon recognition, agonistic antibodies can also be used instead of natural ligands that specifically activate and target the above described receptor Fn 14.
  • the relationship of the functionality of the Fn14 signal pathway and the differentiation of adipocytes found by the experiments of the applicant can be employed for generating new therapeutic substances for treating adiposity/obesity and adiposity-associated diseases.
  • Ligands for PPAR-gamma activate the PPAR-gamma transcription factor that can effect a differentiation of the adipocytes or an activation of the fat metabolism in mature adipocytes.
  • one object of the present invention is to provide substances for the treatment and/or prevention of adiposity/obesity and also for the prevention of adiposity-associated diabetes (type II).
  • the substances comprise agonistic-anti-Fn14 antibodies, antibody derivatives, binding proteins or binding molecules.
  • a specific embodiment of the present invention relates to substances (in particular, agonistic anti-Fn14 antibodies, antibody derivatives, binding proteins or binding molecules) for the treatment and/or prevention of adiposity/obesity and thereby also for the prevention of adiposity/obesity-associated diabetes (type II) that are not associated with any controversial toxicities in effective doses for a relevant therapy.
  • substances in particular, agonistic anti-Fn14 antibodies, antibody derivatives, binding proteins or binding molecules
  • a further object of the present application is to determine the necessary binding sites or epitopes for activation on the receptor and methods and processes based on this previously unavailable knowledge that allow for an efficient and targeted generation of activating antibodies. Based on the gained knowledge about the binding sites or epitopes on the receptor that are necessary for activation it is then a further object of the present application to identify substances, in particular, agonistic anti-Fn14 antibodies, antibody derivatives, binding proteins or binding molecules, that bind to the binding sites or epitopes on the receptor and by doing so activate the receptor signal transduction and to provide means for the treatment and/or prevention of adiposity/obesity and by doing so to also prevent adiposity/obesity-associated diabetes (type II).
  • substances in particular, agonistic anti-Fn14 antibodies, antibody derivatives, binding proteins or binding molecules
  • the above problem of treating and/or preventing adiposity/obesity is solved by generating substances that effectively inhibit the differentiation or function of adipocytes.
  • These substances are characterized according to the invention by their ability to induce the Fn14-mediated signal transduction.
  • agents that induce Fn14-mediated signal transduction are the natural ligands or homologous ligands (or non-species ligands) of Fn14 as well as antibodies that recognize Fn14 at defined positions (epitopes) and activate at least on of these epitopes by binding.
  • the use of antibodies that recognize and activate Fn14 has the advantage that these are easy to prepare and that they can be used in patients in a more controlled manner due to their good pharmacological behaviour.
  • the invention also comprises epitopes on the receptor Fn 14 that also initiate the molecular event of binding that takes place when the natural ligands bind to the receptor and, therefore, leads to receptor activation and subsequent modulation of the differentiation of adipocytes.
  • agonists according to the invention such as antibodies and derivatives thereof, that recognize and activate the receptor Fn 14.
  • agonistic antibodies and compositions are produced in a target manner by techniques and methods that are based on the knowledge about the necessary positions (epitopes) of Fn 14 for receptor activation.
  • These agonistic antibodies and compositions containing these are employed for the treatment of adiposity/obesity and further metabolic diseases that are associated with increased or aberrant amount of fat cells by stimulating the large Fn 14 signal pathway.
  • a preferred embodiment of the invention describes the use of at least one antibody directed against Fn14 (anti-Fn14 ab) for modulating the differentiation of fat cells; the use of a monoclonal or recombinant antibody (anti-Fn14 mab or anti-Fn14 recab) being preferred.
  • a further embodiment of the invention describes antibodies or antibody derivatives that are directed against specific epitopes of the receptor that cause the activation of the receptor by antibody binding.
  • binding molecules for example, affilines, anticalines, aptameres
  • a further embodiment of the invention describes specific epitopes of the receptor that cause activation of the receptor upon binding by antibodies, antibodies derivates, binding proteins or binding molecules.
  • the invention provides a new screening method for selecting Fn 14- activating agents, wherein these agents comprise amongst others anti-Fn14 antibodies, derivates thereof, binding proteins and binding molecules and allow for the activation of the Fn14 by binding to at least one activating epitope of Fn14.
  • the approach used for said purpose employs, for example, antibodies against epitopes of the receptor that cause the activation of the receptor upon antibody binding and initiate the receptor signal pathway. This can either be measured directly by measuring the signal transduction events or indirectly, for example, by analysing the modulation of cellular phenotypes by, for example, inhibiting the differentiation of fat cells.
  • the method used for, for example, testing the agonistic properties and utilities of putative agonistic antibodies for Fn 14 for modulating the differentiation of fat cells is described by reference to examples and preferably comprises the following steps:
  • the cells activated for differentiation in this way are incubated with the agents that are to be tested such as anti-Fn14 antibodies, antibody preparations or formulations.
  • the measurement of the differentiation of cells is done, for example, after > 8 days with a test that is suited for the detection of mature fat cells (for example, with a test for detecting lipids, e.g. Nile Red Assay, that is described in Example 1). 5) Agents that modulate the differentiation of fat cells are recognized because in these cells a deviation of the strength of the test signal occurs that is more than a standard deviation (1 x), preferably more than a 2-fold change of the signal, that is provided by the negative control antibodies.
  • This method that is only listed as an example can be modulated and adapted for the purpose of identifying further Fn 14 activating antibodies that recognize epitopes that cause the activation of the receptor upon binding.
  • the Fn14- activating effect of agents can be determined, for example, by detecting specific signal transduction events in the cell.
  • the anti-Fn14 antibody (or a combination of antibodies) that inhibits or modulates the differentiation of adipocytes is an Fn14-activating agent in this experiment.
  • These agents were used for preparing a pharmaceutical composition for treating adiposity/obesity as well as further metabolic diseases that are associated with increased or aberrant amounts of fat cells.
  • the invention also relates to the use of functional variants of the above listed antibodies for preparing a pharmaceutical composition for the treatment of adiposity/obesity and further metabolic diseases that are associated with an increased or aberrant amount of fat cells.
  • the invention also relates to the use of substances and preparations that have activating epitopes of Fn14 for the direct immunization of patients for preventing and/or treating adiposity/obesity and/or further adiposity/obesity- associated diseases that are associated with an increased or aberrant amount of fat cells.
  • adipocyte differentiation relates to the ability of a correspondingly determined precursor cell (pre-adipocyte) to terminally differentiate upon a suitable stimulus. By doing so a specific gene expression program is initiated that phenotypically leads amongst others to an intracellular accumulation of lipid. Such a differentiated adipocyte is also denoted a mature adipocyte.
  • metabolic syndrome relates the variable combination of clinical parameters and symptoms that are associated inter alia with adiposity/obesity. Amongst others this frequently includes adiposity and/or high blood pressure and/or high cholesterol and/or insulin resistance.
  • epitopope (or antibody binding site) is defined as the spatial structure of an antigen that is recognized and bound by an antibody.
  • Polyclonal antibodies (pab) are mixtures of different antibodies synthesized by different B-cell clones that may recognize different epitopes because they have different antigen binding sites.
  • Fc-domain of an antibody relates to a part of a molecule that comprises the CH2-, CH3- and hinge regions but that lacks the antigen binding sites.
  • Fn14 activating agent relates to any agent that is capable of imitating ligand binding to Fn14, cluster formation of Fn14 on the cell surface or that allows or enhances the Fn14 signal pathway or that can influence how the cell interprets the Fn 14 signal on its inside.
  • Fn14 activating agents are TNFSF12, soluble anti-Fn14 antibodies, cross-linked anti-Fn14 antibodies as well as multivalent anti-Fn14 antibodies.
  • Fn 14 signal pathway relates to all molecular reactions in the context of the ligand-, antibody- or binding partner-mediated activation of Fn 14 and the resulting molecular reactions.
  • anti-Fn14 antibody (“anti-Fn14 ab”) relates to all antibodies that recognize at least one epitope of the Fn14 receptor and bind to it.
  • anti-Fn14 monoclonal antibody (“anti-Fn14 mab”) relates to all monoclonal antibodies that recognize one single epitope on the Fn 14 receptor and bind to it.
  • anti-Fn14 antibodies poly- or monoclonal, cross-linking agent
  • This also includes antibodies that can aggregate the receptors, so that the antibody can bind to the surface of potential target cells and multiply receptor cluster formation there.
  • the term "functional variant of an antibody” relates to an antibody and/or fragment that essentially mediates the biological function or functions of the antibody. In the case of the present antibodies this can be the ability to inhibit the lipid storage in suitable cells.
  • the scope of this term also comprises various derivatives of antibodies, in particular, recombinant, chimeric, humanised or otherwise modified antibodies that induce Fn14 signal transduction.
  • the term "functional variant” according to the invention relates to all "non-antibody” proteins having similar binding properties such as, for example, anticalines, affilines, single domain antibodies and other specific binding proteins.
  • the term "functional variant” of a polypeptide or a nucleic acid preferably relates to polypeptides or nucleic acids having a sequence similarity, in particular, a sequence identity, of at least 25 %, preferably about 40 %, particularly preferred about 60 %, more preferably about 70 %, most preferred about 80 %, in particular about 90 % and very most preferred 98 % to the polypeptide.
  • Said variants are, for example, homologous polypeptides that originate from other organisms.
  • variants are polypeptides or fragments that are coded by the different alleles of a gene.
  • Functional variants preferably also include naturally occurring mutations, in particular, mutations that quantitatively change the activity of the peptides that are coded by these sequences. Furthermore, these variants may preferably result from differential splicing of the coded gene.
  • the term "functional variant” includes derivates having single nucleotide polymorphisms (SNP).
  • sequence identity relates to the degree of identity (% identity) of two sequences that, in the case polypeptides, may be determined, for example, by BLASTP 2.2.10, and in the case of nucleic acids, for example, by BLASTN 2.2.10, wherein the low complexity filter is turned off and, in the case of BLASTP 1 the matrix is BLOSUM 62 (Altschul et al. 1997, Nucleic Acids Res., 25:3389 - 3402).
  • sequence similarity or “sequence homology” relates to the similarity (% positives) of two nucleotide or polypeptide sequences that is determined, for example, by BLASTN 2.2.10 or by BLASTP 2.2.10, wherein the filter is turned off, and in the case of BLASTP the matrix is BLOSUM 62 (Altschul et al. 1997, Nucleic Acids Res., 25:3389 - 3402).
  • binding protein or "binding peptide” according to the invention relates to a class of proteins, peptides or fragments that bind to or inhibit the corresponding molecule including without limitation polyclonal or monoclonal antibodies, antibody fragments and protein scaffolds that are directed against these proteins, peptides or fragments.
  • aptamer describes nucleic acids that bind to a polypeptide with high affinity.
  • Aptamers can be isolated from a large pool of different single-stranded RNA molecules by selection methods such as SELEX (see, e.g., Jayasena, Clin. Chem., 45, pp. 1628 - 1650, (1999); Klug and Famulok, M. MoI. Biol. Rep., 20, pp. 97 - 107, (1994); US 5,582,981).
  • Aptamers can also be synthesized and selected in their mirror form, for example, as the L-ribonucleotide (Nolte et al., Nat.
  • the general method for preparing an antibody or antibody fragment is by methods that are known to the expert, for example, by immunizing a mammal, for example, a rabbit, with the corresponding antigen, whereby, if necessary, corresponding adjuvants, for example, Freund's adjuvant and/or aluminium hydroxide gels or other adjuvants may be used (see, for example, Diamond, B.A. et al., The New England Journal of Medicine, pp. 1344 - 1349, (1981)).
  • the polyclonal antibodies that are formed in the animal as the result of an immunological reaction can later on be isolated from blood by using methods known in the state of the art and may then be purified, for example, by column chromatography.
  • monoclonal antibodies can be prepared in accordance with the known methods of Winter & Milstein (Winter, G. & Milstein, C 1 Nature, 349, pp. 293 - 299, (1991)).
  • Specific polyclonal antibody serums that are directed against the human Fn 14 can be prepared by employing conventional methods by injecting animals, for example, goats, rabbits or mice, subcutaneously, for example, with an Fn14-derived protein or peptide or derivative that presents the activating epitope.
  • animals for example, goats, rabbits or mice
  • DNA vaccination may be employed.
  • the activating natural epitope can be represented by any chemical substance that has a surface structure and/or form and/or charge that is comparable to the natural epitope.
  • intraperitoneal or subcutaneous injections of additional agents that enhance the immune reaction may also be employed.
  • adjuvants e.g. Freund's adjuvant
  • Polyclonal antiserums that contain the desired antibodies that are directed against the activating epitope of Fn 14 can be expanded by repetitive intraperitoneal immunizations of mice with Fn14-derived protein or peptide or derivative in the absence of adjuvants.
  • the immunization of animals with Fn14, derived proteins, peptides or derivatives thereof that represent the activating epitope can also be effected by either intraperitoneal or intravenous injections.
  • hybridoma cells For preparing monoclonal antibodies hybridoma cells can be fused according to classical methods and be screened, for example, by an ELISA (Ling et al., J. Interferon and Cytokine Res., 15, pp. 53 - 59 (1995)). Furthermore, hybridoma cells are assayed for their ability to produce antibodies that recognize the Fn14, derived protein, peptide or derivative or the activating epitope and to modulate the differentiation of fat cells. Pure monoclonal antibodies (IgG) were purified from hybridoma cell culture supematants by means of protein A sepharose.
  • anti-Fn14 antibodies can also be prepared by employing standard methods for producing recombinant DNA (Winter and Milstein, Nature, 349, pp. 293 - 299 (1991)).
  • "chimeric" antibodies wherein the antigen binding site of an animal antibody is coupled to a human constant domain can be prepared (e.g., Cabilly et al., US 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81, pp. 6851 - 6855 (1984)). Chimeric antibodies reduce the observed immune response that becomes pronounced in human clinical studies, where animal antibodies are used.
  • antibody and antibody fragment is also understood to include antibodies and/or antigen binding parts thereof that were produced recombinantly and, if required, were modified, for example, chimeric antibodies, humanized antibodies, multifunctional antibodies, bispecific or oligospecific antibodies, single-stranded antibodies and F(ab)- or F(ab) 2 fragments (see, for example, EP 368 684 B1 , US 4,816,567, US 4,816,397, WO 88/01649, WO 93/06213 or WO 98/24884).
  • Humanized antibodies that recognize the activating epitope of Fn14 can be synthesized.
  • Humanized antibodies are chimeric antibodies that for the largest part have human IgG sequences into which regions responsible for the specific antigen binding have been inserted (WO 94/04679). Animals are immunized with the desired antigen, the corresponding antibody is isolated and that part of the variable sequence regions that is responsible for the specific antigen binding is removed. The antigen binding sites originating from the animals are then cloned into the corresponding position of the human antibody gene, wherein the human antigen binding sites had been deleted. Humanized antibodies reduce the use of heterologous (inter-species) sequences in human antibodies and pose a lower risk for inducing an immune response in the treated individual.
  • anti-Fn14 antibodies that recognize the activating epitope can also be achieved by preparing chimeric or humanized antibodies with anti-Fn14 variable domains and human constant domains (CH1 , CH2, CH3) that had been isolated from different classes of immunoglobulins.
  • anti-Fn14 antibodies that recognize the receptor activating epitope can be recombinantly produced with an increased affinity for antigen binding sites by cloning the antigen binding sites into vectors that contain the corresponding human constant regions (Arulandam et al., J. Exp. Med., 177, pp. 1439 - 1450 (1993); Lane et al., Eur. J. Immunol., 22, pp. 2573 - 2578 (1993); Traunecker et al., Nature, 339, pp. 68 - 70 (1989)).
  • protein scaffolds for example, anticalines, that are based on lipocaline (Beste et al., Proc. Natl. Acad. Sci. USA, 96, pp. 1898 - 1903, (1999)).
  • the natural ligand binding sites of lipocalines, for example, of the retinol-binding protein or bilin-binding protein can be changed, for example, by employing a "combinatorial protein design” approach, and in such a way that they bind selected haptens (Skerra, Biochem. Biophys. Acta, 1482, pp. 337 - 350, (2000)).
  • binding protein is to be understood as also including the herein described binding proteins and binding molecules, e.g. affilines, anticalines and aptameres, that specifically recognize the receptor-activating epitope of the receptor Fn 14, proteins derived from these, peptides or derivatives thereof, that were produced recombinantly, and if required, were modified.
  • Antibodies bind with a defined specificity and affinity to the corresponding target molecules. This also true for antibodies that are directed against receptors on the cell surface.
  • the binding of an antibody to such a surface receptor, here Fn14, can result in its activation. For doing so the mere binding of the antibody is necessary but not sufficient.
  • the receptor activation depends on the type of binding of the antibody. This type of binding of the antibody must simulate the effect that the binding of a natural ligand has on the receptor. In many cases the natural ligand causes a change in conformation or another property of the receptor, for example, its dimerisation or multimerisation or both.
  • an activation results, when these structural changes result in an intrinsic activity of the receptor protein, for example, an enzymatic activity or a protein- protein interaction activity directly or indirectly. Induced changes of the receptor depend on the molecular position and the positions on the receptor, to which the ligand or antibody binds. Molecular positions that are recognized by antibodies are denoted epitopes. Accordingly, antibodies that lead to the activation of receptors bind to those epitopes that simulate the receptor effects of ligand binding (see above) upon binding.
  • peptides, epitopes or artificial chemical structures that correspond functionally to the epitopes can also be used for immunizing animals for the generation of, for example, polyclonal or monoclonal antibodies as well as for generating antibody derivatives and binding proteins by means of recombinant technologies.
  • activating epitopes as immunogen for forming activating antibodies directly in humans. This allows a vaccination of patients whereby their adiposity/obesity and their consequential damages may be prevented and/or treated.
  • the knowledge about the epitopes that lead to receptor activation upon binding can inter alia be employed for generating agonistic antibodies with a targeted rational method. This can amongst others be achieved by immunizing with said epitopes.
  • direct binding assays based on activating epitopes that, for example, are based on the ELISA principle (see Example 5, Figure 6) will also be established and used. Said assays can be employed for identifying and characterizing activating antibodies (see Example 5, Figure 6).
  • the anti-Fn14 antibodies, antibody derivatives or binding proteins thereof used according to the invention function by inhibiting the formation of terminally differentiated adipocytes.
  • the formation and/or function of terminally differentiated adipocytes can be inhibited by inhibiting the differentiation to terminally differentiated adipocytes, in particular, by inhibiting the transcription signal pathway for differentiation.
  • the formation of terminally differentiated adipocytes can be inhibited by inhibiting lipogenesis or by enhancing lipolysis.
  • the inhibition of the differentiation to terminally differentiated adipocytes can be done by measuring the lipid (e.g. by staining with Nile Red).
  • reporter assays such as, e.g., for PPAR- ⁇ or CEBP- ⁇ or - ⁇
  • the inhibition of the transcription signal pathway can be measured.
  • lipophilic coloring agents such as, e.g. Nile Red
  • the lipid content in adipocytes can be detected.
  • the modulation of the differentiation of adipocytes by activating Fn14 is described for the natural receptor ligands TNFSF12 as well as for activating anti-Fn14 antibodies in Example 2.
  • Fn14-mediated signal transduction in inflammatory and toxic processes and cell death (for example, in tumor cells) is already known, it was also analyzed, if the inhibition of the differentiation of adipocytes is associated with the induction of cell death.
  • the determination of the cell count of living cells is possible, for example, with the "Alamar Blue” (Biosource) or “CellTiterGlo” (Promega) assays.
  • the invention relates to a method for the treatment of a patient, wherein an effective amount of one and/or more of the above defined agonistic agents such as anti-Fn14 antibodies is administered to the patient.
  • the patient Preferably, the patient suffers from adiposity/ obesity.
  • all of the embodiments illustrated above for the use according to the invention apply, too.
  • the factors TNFSF12 and the anti-Fn14 antibodies found according to the invention allow for the inhibition of the differentiation of pre-adipocytes to adipocytes and/or the inhibition of the function of mature or maturing adipocytes, for example, by inhibiting the lipogenesis or assisting lipolysis.
  • the inhibiting function is the differentiation of adipocytes in patients with adiposity/obesity or the prevention of adiposity/obesity.
  • the term "inhibitor” describes an agent, such as an anti-Fn14 antibody, that preferably inhibits the process of fat storage by itself.
  • the activating antibody can be denoted an inhibitor.
  • the inhibition can, for example, be achieved by the induction of the Fn14-dependent signal transduction and subsequent modulation of the expression of regulated specific genes.
  • the inhibitor used e.g., the anti-Fn14 antibody
  • this can take place by interfering with the differentiation to terminally differentiated adipocytes, in particular, by modulating the Fn 14 transcription signal pathway for the differentiation in pre-adipocytes.
  • the invention relates to a pharmaceutical composition that contains one or more agents of the present invention. For all the pharmaceutical compositions according to the present invention the following holds true:
  • the corresponding molecules are typically formulated together with suitable additives or excipients, such as, for example, physiological buffer solution, for example, sodium chloride solution, deionised water, stabilizers, such as protease- or nuclease inhibitors, preferably aprotinin, ⁇ -aminocaproic acid or pepstatin A or chelators, such as EDTA, gel formulations, such as white vaseline, paraffin with low viscosity or yellow wax, depending on the mode of the administration.
  • suitable additives or excipients such as, for example, physiological buffer solution, for example, sodium chloride solution, deionised water, stabilizers, such as protease- or nuclease inhibitors, preferably aprotinin, ⁇ -aminocaproic acid or pepstatin A or chelators, such as EDTA
  • suitable additives or excipients such as, for example, physiological buffer solution, for example, sodium chloride solution, deionised water, stabilizers,
  • detergents such as, for example, Triton X-100 or sodium deoxycholate
  • polyols such as, e.g., polyethylene glycol or glycerin
  • sugars such as, for example, sucrose or glucose
  • amphoteric substances such as amino acids, such as glycine or, in particular, taurin or betaine
  • a protein such as, for example, bovine or human serum albumin.
  • Detergents, polyols and/or amphoteric substances are preferred.
  • the physiological buffer solution has a pH of about 6.0 - 8.0, in particular a pH of about 6.8 - 7.8, in particular a pH of about 7.4, and/or an osmolarity of about 200 - 400 milliosmol/Iiter, preferably of about 290 - 310 milliosmol/liter.
  • the pH of the medicaments is generally adapted by employing a suitable organic or inorganic buffer, such as, for example, phosphate buffers, tris buffers (tris(hydroxy methyl)amino methane), HEPES buffer ([4-(2-hydroxy ethyl)piperazino]ethane sulfonic acid) or MOPS buffer (3- morpholino-1 -propane sulfonic acid).
  • a suitable organic or inorganic buffer such as, for example, phosphate buffers, tris buffers (tris(hydroxy methyl)amino methane), HEPES buffer ([4-(2-hydroxy ethyl)piperazino]ethane sulfonic acid) or MOPS buffer (3- morpholino-1 -propane sulfonic acid).
  • phosphate buffers tris buffers (tris(hydroxy methyl)amino methane)
  • HEPES buffer [4-(2-hydroxy ethyl)piperazino
  • Solutions for injection are generally employed, when only a small amount of a solution or suspension, for example, about 1 to about 20 ml is to be administered.
  • Solutions for infusions are generally employed, if a larger amount of a solution or suspension, for example, one or several litres, is to be administered. Because contrary to an infusion solution only a few millilitres are administered in the case of solutions for injection, small differences in the pH or the osmotic pressure of the blood or the tissue liquid are not perceived in comparison to a solution for injection or do not play an important role. Therefore, the dilution of the formulation before its use is generally not necessary. However, when relatively large amounts are to be administered, the formulation according to the invention should be diluted shortly prior to the administration, so that an isotonic solution is obtained. An example of an isotonic solution is a 0.9% saline solution.
  • the effective dose will depend on the weight and condition of the subject to be treated. It is to be assumed that the skilled person knows how to determine a suitable dose.
  • the pharmaceutical composition can be administered in various modes and ways, for example, intramuscularly, subcutaneously, intrathecal ⁇ , into the fat tissue, percutaneously (dissolved in DMSO), intravenously or intraperitoneal ⁇ or by fusion or gels, that contain the respective medicament. Further, it is possible to apply the medicament topically and locally. Furthermore, the medicament can be administered by a transdermal therapeutic system (TTS) that allows for the time-controlled release of the medicament.
  • TTS transdermal therapeutic system
  • TTS are known from EP 944 398, EP 916 366, EP 889 723 or EP 852 493.
  • a slower release of the protein, peptide or antibody is achieved by a combination with polymers, a prolonged half life by the addition of PEG.
  • Suitable polymers also allow for the oral administration of the pharmaceutical composition, for example, by providing a protected passage through the colon and the targeted penetration of the cell-cell contact sites in the area of the colon epithelium.
  • a combination with chemical active agents e.g. appetite inhibitors, lipase inhibitors
  • the pharmaceutical composition according to the invention having an activating agent is provided in a form that allows for the contact of the active agent with a component of the cell surface (does not enter the cell) (Cardiovasc. Pharmacol. Ther., 7 (3) pp, 171 - 80, (July 2002)).
  • the invention relates to a method for the prevention of adiposity/obesity and/or the treatment of a patient, wherein an effective amount of one or more of the above defined inhibitors is administered to the patient.
  • the patient suffers from adiposity/obesity or diabetes type Il or from diabetes type ll-related diseases.
  • the soluble polypeptides and antibodies employed according to the invention are capable of inhibiting the storage of lipids in murine 3T3-L1 cells and in human pre-adipocytes.
  • the inhibition was demonstrated in primary human adipocytes of subcutaneous as well as of omental origin. In each case the detection was done by the Nile Red assay.
  • the receptor Fn 14 the activation of which effects the modulation of the differentiation of fat cells, is present on human and murine precursor fat cells. This fact is an important prerequisite for explaining the observed phenotype by receptor-mediated signal transduction.
  • Fig. 1 This illustrates the dose/response curve that shows the lipid content of 3T3-L1 cells in dependency of the added amount of a recombinant, secreted or soluble protein fragment of TNFSF12. The measured results are specified as relative fluorescence units (RFU) on day 5 after the addition of the protein.
  • REU relative fluorescence units
  • the primary human cell cultures were incubated with each of the respective recombinant proteins.
  • the percentage data relates to control cultures that were treated with a protein concentration of 0.025 ng/ml that is still ineffective.
  • a low signal strength means a low lipid storage in the differentiating human adipocytes, a strong signal indicates a strong lipid storage.
  • Fig. 3 Detection of Fn 14 on murine and human pre-adipocytes
  • A This illustrates an analysis of the competition of the binding of the Fn14-specific antibody mAB-X1 by the peptide with SEQ-ID #4.
  • a positive control a fusion protein containing the complete human Fn 14 extracellular domain was employed and a control peptide as a negative control.
  • the fluorescence intensity distributions for HeLa cells are specified for each competition experiment obtained with the specific antibody (with competitor - full line or without the competitor - closely dashed line) or an isotype control antibody instead of the Fn14-specific antibody (barred dashed line).
  • the fluorescence intensity distribution after the addition of the competitor is also labelled with an arrow at the maximum.
  • a shift of the fluorescence distribution to the left with the competitor (full line) relative to the distribution without competitor (closely dashed line) illustrates its competitive binding to an Fn14-specific antibody.
  • Fig. 7 This illustrates the limitation of the gain in body weight in male C57BL/6 mice two weeks after injection of a suitable expression vector that is dependent on the extent of the expression of soluble or secreted protein fragment of murine TNFSF12(A). The extent of the expression of the protein fragments was determined on serum samples by an ELISA. The negative correlation of the protein expression and the gain in body weight is illustrated.
  • Example 1 Example 1:
  • 3T3-L1 cells are mouse fibroblast cells of embryonic origin that can differentiate terminally to mature fat cells (adipocytes) under suitable conditions. Therefore, the 3T3-L1 kept in proliferation culture are also called pre-adipocytes. They grow in monolayers and have a doubling time of about 24 h.
  • the 3T3-L1 cells used herein originate from ATCC (CL-173). Because the 3T3-L1 cells spontaneously differentiate at confluency and, thereby, change the properties of the culture, they were not cultivated until confluency (max. 80%). The passaging was done at intervals of 2 days without a medium exchange in between. The cells were used from passage 5 on and until passage 18 only.
  • the maintenance culture of the 3T3-L1 cells was kept in T-75 culture flasks. For passaging every 2 days these were seeded with 4.5 x 10 5 cells/T-75 flask. There is no medium exchange until the next passage.
  • the seeding of the 3T3-L1 cells for the next experiment was conducted in 96-well microtiter plates: The 3T3-L1 cells were seeded at a cell density of 1.5 x 10 4 cells/well in 200 ⁇ l growth medium/well. Then the cells were incubated without medium exchange for 3 days and subsequently induced for differentiation as it is described further below and contacted with recombinant soluble or secreted protein fragment or antibody.
  • the 3T3-L1 growth medium had the following composition: DMEM, 10% FCS, 1% penicillin-streptomycin, 2% glutamine, 1% Na-pyruvate).
  • the differentiation medium was principally added as a double concentrate directly after the addition of recombinant protein or antibody. In this form it consists of a 3T3-L1 growth medium supplemented by induction factors (200 nM insulin, 1 mM IBMX und 2 ⁇ M dexamethasone).
  • pre-adipocytes The isolation and differentiation of pre-adipocytes was conducted as described in Hauner et al. [Methods MoI. Biol. 2001; 155: 239 - 47].
  • the fat tissue obtained during surgical operations is mechanically freed of connective tissue and remaining blood vessels and subsequently minced. Then it is digested with 200 U/ml collagenase NB4 (Serva Electrophoresis GmbH, Heidelberg) for 90 min at 37 0 C and 80 rpm in PBS with 2 % BSA (3 ml collagenase solution/g fat tissue). Afterwards, centrifugation followed for 10 min at 200 g.
  • the formed pellet also contained erythrocytes and remaining connective tissue next to the pre-adipocytes and was taken up in erythrocyte lysis buffer (155 mM NH 4 CI, 5.7 mM K 2 HPO 4 , 0.1 mM EDTA, pH 7.3). After a maximum incubation of 10 min at room temperature it was filtered over a 150 ⁇ M filter and centrifuged as described above.
  • the remaining pellet still contained remaining connective tissue, it was again purified with a 70 ⁇ M filter after being taken up in pre-adipocyte medium (DMEM/F12 (Invitrogen GmbH, Düsseldorf) supplemented with 8 mg/l biotin, 4 mg/l pantothenate, 1.79 g/l NaHCO 3 and 55 mg/l pyruvate).
  • DMEM/F12 Invitrogen GmbH, Düsseldorf
  • the cells were counted, centrifuged again and resuspe ⁇ ded in DMEM/F12 with 10 % FCS and 50 ⁇ g/ml gentamycin.
  • the seeding was done at a density of 40000 to 55000 cells/cm 2 .
  • the cells were incubated at 37 0 C and 5 % CO 2 .
  • 3T3-L1 cells Three days after seeding the 3T3-L1 cells 140 ⁇ l medium of the 3T3-L1 cells were taken and 50 ⁇ l growth medium was added, wherein suitable amounts of recombinant protein (0 - 3 ⁇ g/ml protein) or antibody (for example, 0 - 10 ⁇ g/ml) had been dissolved. In addition, 100 ⁇ l 2X differentiation medium was added to the 3T3-L1 cells followed by an incubation of the 3T3-L1 cells in an incubator for 5 days. After this time the intracellular incorporated lipids were determined.
  • the cells were washed twice with PBS and then incubated in serum-free differentiation medium (pre-adipocyte medium with 66 nM insulin, 1 nM T 3 , 100 nM hydrocortisone, 10 ⁇ g/ml transferrin, 50 ⁇ g/ml gentamycin). Also, for the first 3 days 1 ⁇ g/ml troglitazone and 0,5 mM IBMX were added. After this time and in the further course the medium was exchanged every three to four days using differentiation medium. The cells were used, when at least 50% of the cells (relative to the cell count) had incorporated fat. Measurement of the intracellular stored lipid:
  • Nile Red reagent (Molecular Probes, Leiden, Netherlands; CAS number 7385-67-3) (Nile Red staining solution: 4 ⁇ g/ml Nile Red in PBS/40 % DMSO) was used. The fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 590 nm. The required amount of Nile Red was added to the calculated amount of DMSO and mixed. Subsequently, the calculated amount of PBS is added and the solution is mixed.
  • REU relative fluorescence units
  • the activation of a cellular receptor can be done by the natural ligand as well as by an antibody, when the activation of said receptor is effected, for example, by successful cluster formation of receptor molecules, which is the present case.
  • the antibody functions agonistically when it results in a similar activity after binding to its target structure that is also generated by the natural ligand. When doing this, the affinity may deviate.
  • the epitope of the antibody can, but must not necessarily, correspond to the binding site of the ligand. What is important is the functional effect on the activation status of the receptor. Not any binding antibody will also result in an activation of the receptor, but only those that bind to specific epitopes in the extracellular domains of the receptor that will result in such an activation.
  • a receptor was activated by its natural ligand.
  • the human ligand was also capable of activating the corresponding homologous mouse receptor.
  • the inhibition of lipid storage in mouse 3T3-L1 cells (Fig. 1) or in primary human adipocytes (Fig. 2) was determined with a recombinant, secreted or soluble protein fragment of human TNFSF12 (R&D Systems GmbH, Wiesbaden: Cat No.: 1090-TW; amino terminal 6X HIS-tagged extracellular domain of human TWEAK (arg 93 - his 249, see Chicheportiche et al., 1997, J. Biol. Chem. 272:32401 - 32410)).
  • the lipid storage can serve as a measure for the differentiation of adipocytes or as a measure for the regulation of lipogenesis or lipolysis in these cells.
  • a comparable inhibition of differentiation was achieved for primary fat cells of omental origin and the human SGBS adipocyte cell line.
  • the inhibition of the storage of lipids in primary human adipocytes was determined with the antibody mAB-X1 (Fig. 4).
  • the results demonstrate that the storage of lipids in primary human adipocytes (Fig. 4) during the differentiation was inhibited in a dose- dependent manner by adding the antibody mAB-X1.
  • the storage of lipids can serve as a measure for the differentiation of the adipocytes or as a measure for the regulation of lipogenesis or lipolysis in these cells.
  • the addition of 1 ⁇ l mAB-X1 antibodies in this experiment led to an inhibition of differentiation that was comparable to the case when 100 ng/ml naturally ligand had been employed.
  • the cells were subsequently measured in the FACS device. For doing this the measurement of the fluorescence intensities was done for living cells only.
  • the result demonstrates that the receptor described for TNFSF12 is expressed on the cell surface of 3T3-L1 cells as well as on the surface of primary human pre-adipocytes.
  • the mode and manner how the proteins according to the invention accomplish the inhibition of lipid storage in adipocytes was determined in more detail by further analysis of the cell cultures.
  • An inhibition of the differentiation or the lipid storage in differentiating adipocytes can, for example, be effected by protein-mediated activities such as cell death, preventing the exit from the cell cycle, blocking a differentiation- specific gene expression or blocking lipogenesis or by enhancing lipolysis.
  • Figure 9 illustrates the determination of the cell count of subconfluent 3T3 cell cultures.
  • the cells were seeded at a density of 3000 cells per well in a 96-well cell culture dish. 24 h later the growth medium of the 3T3-L1 cells was exchanged against medium with reduced FCS content (0.5%). Another 48 h later a further medium exchange to 2 % FCS and a further addition of soluble recombinant TNFSF12 was done. In the course a dose- response relationship was investigated over a concentration range of 0.1 to 500 ng/ml protein.
  • mice of the strain C57/BI6/J Following the determination of the maximum tolerated dose (MTD) in mice of the strain C57/BI6/J a) the reduction of the gain in weight in normal weight mice (without previous high calorie diet) and b) the net reduction of adipose tissue in mice that were normal weight or obese at the beginning of the test (and initiating or maintaining a high calorie diet) after addition of the proteins used according to the invention were assayed by weighing or determining the BMI ("body mass index").
  • MTD maximum tolerated dose
  • the determination of the maximum tolerated dose was done with 3 mice (strain C57/BI6/J wild type) by administering 0.002; 0.02; 0.2; 2.0 and 10 mg protein/kg as a one-time intravenous dose.
  • a subsequent fine adjustment of the dose was done with 3 mice each (strain C57/BI6/J wild type) by the one-time addition of the protein.
  • the dose necessary for application is determined by the repetitive administration of a selected dose over several applications based on blood parameters and serum stability.
  • the stimulation was done with 10 '7 , without or 10 "10 insulin, because 10 '10 insulin is not stimulating in this context.
  • 10 '10 insulin is not stimulating in this context.
  • medium DMEM/Ham F12 with 5 mM glucose.
  • Differentiated cells were washed four times with 2 ml warm PBS.
  • 800 ⁇ l DMEM/F12 with 5 mM glucose and each of the proteins according to the invention were added. Subsequently, there was an incubation for 1 h at 37 0 C. Subsequent addition of insulin. Plates were incubated for 15 min at 37 0 C at the lowest agitation frequency.
  • 2-Deoxy-D-[1-H 3 ]-glucose (AmershamTRK 383) was diluted 1 : 10 in DMEM/F12. During the incubation period tubes were provided for the scintillation counter: one for each measuring point and an additional one for the empty well and one for the complete amount of used 2-deoxy-D-[1-H 3 ]-glucose. After 15 min incubation 8 ⁇ l of the diluted 2-deoxy-D-[1-H 3 ]-glucose were added. The plates were again incubated with light agitation for 20 min at 37 0 C. The plates were placed on ice after 20 min. The medium was suctioned off by a pump.
  • TNFSF12 The receptor activation by TNFSF12 in this experimental set up demonstrated no negative influence of the insulin-dependent glucose uptake.
  • Preferred animal models are those that correspond to the human situation of adiposity/obesity or type Il diabetes in a specific manner.
  • rodents such as, for example, mice or rats, as well as dogs, pigs and primates are used.
  • a particularly preferred experimental embodiment is a diet-induced adiposity, optionally associated with an insulin resistance or type Il diabetes in mice or rats induced by high calorie diet.
  • the high calorie diet that is, for example, a 20 - 45 kcal% fat diet, can be administered before or simultaneously to the administration of the proteins or fragments or the pharmaceutical composition of the invention. Accordingly, the animals can be of normal weight or already overweight at the time point of administering the protein or protein fragment.
  • the goal is a reduction or prevention of a gain in weight and/or a weight reduction; optionally, also an enhancement of the insulin-sensitivity or the glucose tolerance. This can either be done during the initiation of or continued high calorie diet or during a change to normal or hypocaloric diet - in the case of already overweight animals.
  • the change of the diets is initiated at the time of the first administration of the protein or protein fragment or the pharmaceutical composition.
  • mice of the strain C57/BI6/J After the determination of the maximum tolerated dose (MTD) in mice of the strain C57/BI6/J (MTD) a) the reduction of the gain in weight in normal weight mice (without previous high calorie diet) and b) the net reduction of the adipose tissue in mice that were normal weight or obese at the beginning of the test (at the beginning or when maintaining the high calorie diet) after the addition of the proteins was determined by weighing or determining the BMI ("body mass index").
  • MMI body mass index
  • the determination of the maximum tolerated dose is done with 3 mice (strain C57/BI6/J wild type) by administering 0.002, 0.02, 0.2, 2.0 and 10 mg protein/kg as a one-time intravenous dose.
  • a subsequent fine adjustment of the doses is done with 3 mice each (strain C57/BI6/J wild type) by the one-time administration of the protein.
  • the dose necessary for application is determined by repetitive administration of a selected dose over several applications based on blood parameters and serum stability.
  • the administered antibodies are, in principle, detectable by ELISA in blood serum. A reduction of the amount of detectable protein fragment in the blood serum over time is assumed, because this is expected for a protein distributing itself in an organism.
  • the inhibitory activity in the mouse serums can be measured by the addition into the 3T3-L1 cell culture assay by Nile Red lipid determination. In this way it can be demonstrated that the antibodies can be administered intravenously and without causing clinical toxicity.
  • the above-determined maximum dose is reduced to the lowest dose that still has a therapeutic effect.
  • said determined dose is administered parenterally (intravenously, intraperitoneal ⁇ , subcutaneously or intramuscularly).
  • the intraperitoneal, intravenous or subcutaneous application is preferred.
  • normal weight mice are kept on a normal calorie diet (at most 20 kcal% fat diet) or on a high calorie diet (45 kcal%) either without (untreated) or with the addition of the proteins according to the invention (treated) for 10 to 14 weeks.
  • the gain in weight is determined by weighing and/or determining the BMI.
  • obese mice For determining the decrease of the gain in weight in obese mice, obese mice (that had been kept on a high calorie diet (45 kcal% fat diet)) were kept on a normal calorie diet (at most 20 kcal% fat diet) and/or on a continued high calorie diet either without (untreated) and with the addition of the proteins according to the invention (treated) for 10 to 14 weeks. During or after these experiments the gain in weight was determined by weighing and/or determining the BMI.
  • mice 8 five week old male C57BL6/J mice were each injected with an expression construct for recombinant secreted and soluble murine protein fragment of TNFSF12. It was the purpose of this experiment to observe an influence of the protein expression on the gain in body weight. The animals were kept on a normal calorie diet for the complete observation period.
  • TNFSF12 expression construct was used that contained a fragment of the mouse homologue that is described further below. This fragment coded for a recombinant soluble, secreted murine form of TNFSF12.
  • the expression constructs were prepared by performing an RT-PCR with a suitable murine RNA source (TNFSF12: RNA from 3T3-L1 cells) and with suitable primers (comprising the sequences coding for: mouse-TNFSF12: Arg105 - His249).
  • TNFSF12 murine RNA source
  • primers comprising the sequences coding for: mouse-TNFSF12: Arg105 - His249.
  • 6 histidine residues at the 5'-end and 2x TGA stop codons and sequences for the restriction cleavage sites Xhol and BgI Il at the 3'end were introduced.
  • the reaction product was inserted directly into the vector pSECTag/FRT ⁇ /5-His-TOPO (Invitrogen, Düsseldorf, Germany) according to the instructions of the manufacturer for the cloning of PCR products.
  • the secretion signal of the protein for the Ig-kappa chain precedes the protein fragment of TNFSF12 aminoterminally in the reading frame followed by 6 histidine residues. Therefore, the open reading frame generated in this manner coded for the following secreted proteins:
  • the reading frame was cloned into the target vector pBS-HCRHPI-A (Miao et al, 2003, Human Gene Therapy 14:1297 - 1305) that was intended for the in vivo gene expression in animals:
  • the excision from the pSECTag/FRT ⁇ /5-His-TOPO vectors was effected with the assistance of the restriction cleavage sites Nhel and BgIII and filling the overlapping ends - introduction into pBS-HCRHPI-A was done by opening with the restriction enzyme EcoRV and ligating the blunt ends with each of the introduced fragments.
  • the resulting plasmid was designated pXAPOI (mTNFSF12).
  • the transfection of the plasmids in C57BL6/J mice coding for recombinant secreted and soluble murine protein fragment of TNFSF12 was done according to a method that was originally described by Zhang et al. (Gene Therapy 2000; 7:1344).
  • the entry of the plasmid DNA, preferably into liver tissue, is achieved by hydrodynamic pressure that is caused by the fast injection of the plasmid-containing solution into the tail vein. There, a gene expression can be realized for weeks up to months.
  • ELISA antibody pairs for murine TNFSF12 For coating: anti-mTWEAK antibody (R&D Systems, Wiesbaden, Germany, cat. no. # AF1237), detection antibody: biotin-conjugated anti-mTWEAK antibody (e-bioscience, clone MTW-1 , cat. no. # 13-9913). The correlation of protein expression and body weight was investigated.
  • Fig. 7A The correlation coefficient indicates how strict a correlation is. Values between -1 and +1 are possible. A value of 0 means that there is no correlation. +1 or -1 indicate the presence of an absolutely strict (for minus: negative) correlation. These correlations were observed over the complete observation period of 8 weeks.
  • Fig. 7 The trend indicated by the correlation was also observed (Fig. 7) by weighing selected fat tissue deposits (..epidydimal fat pads" - defined well-measurable fat tissue at the gonads of male animals, perirenal and colon-associated fat tissue). The reduction of the body weight corresponded to the reduction of the weight of selected fat deposits.
  • Antibodies that are directed against receptors on the cell surface bind with defined specificity and affinity.
  • the binding of an antibody to Fn 14 can cause its activation. For doing so the mere binding of the antibody is necessary, however, not sufficient, because receptor activation depends on the type of binding of the antibody.
  • This type of binding by the antibody must simulate the effect that a natural ligand has on the receptor upon binding.
  • the natural ligand causes a change in conformation or another property of the receptor such as, for example, its dimerisation or multimerisation or both.
  • An activation takes places when these structural changes leads to an instrinsic activity of the receptor protein, for example, an enzyme activity or protein-protein interaction activity directly or indirectly.
  • activating epitope antibodies that lead to the activation of receptors bind to those epitopes (here denoted "activating epitope”) that simulate upon binding the receptor effects of the ligand binding.
  • activating epitope antibodies that lead to the activation of receptors bind to those epitopes (here denoted "activating epitope") that simulate upon binding the receptor effects of the ligand binding.
  • the peptide of the sequence MDCASCRARPHSDFC is such an activating epitope of the receptor Fn14.
  • Antibodies binding to said peptide are capable of activating Fn14.
  • Antibodies that demonstrate a markedly enhanced binding to the activating epitope in comparison to the binding to the control peptide are defined as "binders" of the activating epitope.
  • a further control in these experiments is the analysis of the specificity of the binding to the activating epitope. In order to ensure this, the binding of a further irrelevant antibody to the activating epitope as well as to a control peptide is analyzed. These antibodies must bind to the activating epitope much weaker in comparison to antibodies that recognize the activating epitope.
  • Figure 6 illustrates an example for the identification of an antibody that binds specifically to the activating epitope of Fn14 that is represented by the peptide of the sequence MDCASCRARPHSDFC.
  • 0.02, 0.2, 1 mg/ml of the peptide MDCASCRARPHSDFC (activating epitope) or a control peptide are placed on microtiter plates (MaxiSorb Plate) by incubating for 15 h at 4 0 C in carbonate buffer (0.1 M carbonate buffer, 8.4 g NaHCO 3 and 3.56 g Na 2 CO 3 per I; pH 9.5).
  • the specifically bound test- or control antibody was determined by the addition of the substrate TMB (tetra methyl benzidine, Sigma) and the subsequent incubation for 30 min at RT. After ending the enzymatic reaction with 50 ⁇ l 1 M HCI the strength of the enzyme-dependent staining reaction was determined in an ELISA reader by determining the absorption at 450 nm. The results of such an ELISA analysis are shown in Figure 6. It is clearly recognizable that the antibody mAB-X1 has a markedly enhanced binding to the activating epitope in comparison to the binding to the control peptide (i.e.
  • mAB X1 is clearly identified as a binder of the activating epitope. Furthermore, the analysis of the binding of a control antibody shows that this binding and the used test are specific. The control antibody shows a low binding to the control peptide and to the peptide that represents the activating epitope. Antibodies according to the invention are preferably identified by this ELISA method.
  • FIG. 5 shows the results of these competition experiments.
  • the activating epitope in this experiment is again represented by the peptide with the sequence MDCASCRARPHSDFC.
  • 1.3 x 10 5 HeLa cells that carry the receptor on their surface received 200 ⁇ l PBA with 50 ⁇ l of the antibody peptide (protein) mixture, incubation for 30 min on ice to allow for antibody binding, then pelleting, the supernatant is removed and the cell pellet is washed with 200 ⁇ l PBA to remove unbound antibody. Then, AlexaFluor488 conjugated goat-anti-mouse antibody (Co. Molecular Probes) was added in a concentration of 5 ⁇ g/ml, incubation for 30 min on ice, then the cells were washed twice with 200 ⁇ l PBA and then the number of antibody-bound cells as well as the strength of the binding to these cells was determined by FACS analysis. For doing this, the cells were measured in a volume of 200 ⁇ l in the FACScalibur (Becton Dickinson) in the FL1-H canal without using "gates".
  • FACScalibur Becton Dickinson
  • mice were immunized with a peptide with the sequence MDCASCRARPHSDFC or with a control peptide comprised of a scrambled version of the above sequence. Immunizations were performed at days 0, 14 and 28. Bleedings were taken at days 0 (pre-immune serum) and on days 38, 66 and final bleeding on day 87.
  • the serum from the immunized mice is able to inhibit accumulation of lipid and differentiation of the 3T3-L1 pre-adipocytes to mature adipocytes, while both the no-serum control and the serum from the same mice prior to immunization with the peptide comprised of the epitope sequence MDCASCRARPHSDFC show no effect on fat cell differentiation and lipid accumulation.
  • the epitope sequence claimed in this invention is able to produce agonistic antibodies directed against the Fn 14 receptor and that the serum containing these antibodies is able to inhibit adipocyte differentiation.
  • this approach outlines the feasibility of using peptides containing this epitope sequence for vaccination studies and therapies for the treatment of obesity.

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Abstract

L'invention concerne des agonistes du récepteur Fn14, ainsi que leur utilisation pour activer la voie de signalisation du récepteur Fn14, pour perturber et/ou moduler la différenciation des adipocytes, et pour prévenir et/ou traiter une adiposité/obésité et/ou un syndrome métabolique. Cette invention concerne en particulier des agonistes, de préférence des anticorps qui inhibent la différenciation de pré-adipocytes vers des adipocytes mûrs, et/ou le stockage de molécules lipidiques chez des adipocytes en cours de différenciation ou des adipocytes mûrs, par une transduction de signaux dont la médiation est assurée par ledit récepteur.
PCT/EP2006/004974 2005-05-24 2006-05-24 Anticorps de type agonistes qui se lient au recepteur tweak fn14 et modulent par consequent les phenotypes associes a l'adiposite, et leur utilisation a des fins therapeutiques WO2006125632A2 (fr)

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WO2009140177A2 (fr) * 2008-05-15 2009-11-19 Biogen Idec Ma Inc. Anticorps anti-fn14 et leurs utilisation
US7731963B2 (en) 2000-09-14 2010-06-08 Biogen Idec Ma Inc. TWEAK receptor agonists as anti-angiogenic agents
EP2391385A1 (fr) * 2009-01-30 2011-12-07 Biogen Idec MA Inc. Procédés de régénérescence de tissu pancréatique
US9775899B2 (en) 2005-02-17 2017-10-03 Biogen Ma Inc. Treating neurological disorders
CN112513099A (zh) * 2018-05-31 2021-03-16 香港理工大学 用于癌症、肥胖症、代谢紊乱和相关并发症及合并症的精氨酸耗竭剂的组合物和应用
WO2023232631A1 (fr) * 2022-06-01 2023-12-07 Julius-Maximilians-Universität Würzburg Agonistes imitant tweak liés à une membrane et solubles à base d'anticorps ayant une activité indépendante de fcyr

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7731963B2 (en) 2000-09-14 2010-06-08 Biogen Idec Ma Inc. TWEAK receptor agonists as anti-angiogenic agents
US9775899B2 (en) 2005-02-17 2017-10-03 Biogen Ma Inc. Treating neurological disorders
US9056908B2 (en) 2007-08-03 2015-06-16 Abbvie Biotherapeutics Inc. Therapeutic use of anti-tweak receptor antibodies
EP2545938A1 (fr) * 2007-08-03 2013-01-16 Abbott Biotherapeutics Corp. Utilisation thérapeutique dýanticorps de récepteur anti-TWEAK
WO2009020933A2 (fr) * 2007-08-03 2009-02-12 Facet Biotech Corporation Utilisation thérapeutique anticorps de récepteur anti-tweak
WO2009020933A3 (fr) * 2007-08-03 2009-03-26 Pdl Biopharma Inc Utilisation thérapeutique anticorps de récepteur anti-tweak
WO2009140177A2 (fr) * 2008-05-15 2009-11-19 Biogen Idec Ma Inc. Anticorps anti-fn14 et leurs utilisation
WO2009140177A3 (fr) * 2008-05-15 2010-08-26 Biogen Idec Ma Inc. Anticorps anti-fn14 et leurs utilisation
EP2391385A1 (fr) * 2009-01-30 2011-12-07 Biogen Idec MA Inc. Procédés de régénérescence de tissu pancréatique
JP2012516712A (ja) * 2009-01-30 2012-07-26 バイオジェン・アイデック・エムエイ・インコーポレイテッド 膵臓組織再生のための方法
EP2391385A4 (fr) * 2009-01-30 2013-05-01 Biogen Idec Inc Procédés de régénérescence de tissu pancréatique
CN112513099A (zh) * 2018-05-31 2021-03-16 香港理工大学 用于癌症、肥胖症、代谢紊乱和相关并发症及合并症的精氨酸耗竭剂的组合物和应用
WO2023232631A1 (fr) * 2022-06-01 2023-12-07 Julius-Maximilians-Universität Würzburg Agonistes imitant tweak liés à une membrane et solubles à base d'anticorps ayant une activité indépendante de fcyr

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