WO2006114284A2 - AGONISTIC ANTIBODIES THAT BIND TO THE LT-β-RECEPTOR AND THEREBY MODULATE ADIPOSITY-ASSOCIATED PHENOTYPES AS WELL AS THEIR USE IN THERAPY - Google Patents

Abstract

The present invention relates to agonists of the LT-β-receptor as well as their use for activating the signal pathway of the lymphotoxin-β-receptor for interfering and/or modulating the differentiation of adipocytes and for use in adiposity/obesity and/or metabolic syndrome. In particular, the invention relates to agonists, preferably antibodies that inhibit the differentiation of pre-adipocytes to mature adipocytes and/or the storage of lipid molecules in differentiating or mature adipocytes by receptor-mediated signal transduction.

Description

Agonistic antibodies that bind to the LT-β-receptor and thereby modulate adiposity-associated phenotypes as well as their use in therapy

Technical description of the invention

The invention relates to agonists of the LT-β-receptor as well as their use for activating the signal pathway of the lymphotoxin-β-receptor for interfering and/or modulating the differentiation of adipocytes and for use in adiposity/obesity and/or metabolic syndrome. In particular, the invention relates to agonists, preferably antibodies, that inhibit the differentiation of pre-adipocytes to mature adipocytes and/or the storage of lipid molecules in differentiating or mature adipocytes by receptor-mediated signal transduction. Also, the invention relates to activating epitopes on lymphotoxin-β- receptors, wherein the interaction of these with an agonist such as an antibody causes the receptor to initiate signal transduction. In particular, the invention relates to antibodies that are directed against the lymphotoxin-β-receptor and that function as activating agents for the lymphotoxin-β-receptor. Preferably, this activation can take place alone or also in combination with other lymphotoxin-β-receptor-activating agents (agonists). Furthermore, the invention relates to pharmaceutical compositions and their use for activating the signal pathway of the lymphotoxin-β receptor for interfering and/or modulating the differentiation of adipocytes, in particular for treating adiposity.

Background of the invention

Presently, 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. According to recent understanding adiposity is a chronic health dysfunction that is based on a polygenetic predisposition and aiso caused by environmental factors and is accompanied by a high accompanying and follow-up morbidity. In this context 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.

The presence of obesity or adiposity can be classified as overweight or (clinical) adiposity in its closer context. It can be defined by the so-called "body mass index" (BMI). The BMI describes the ratio of the body's weight and the body^'s size. The BMI is calculated according to the following formula: BMI = body weight (kg) divided by body size to the square (m²).

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/ m²) lies in the range with the lowest relative mortality risk. The BMI values 25 and 30 kg/ m² are the corresponding risk- oriented limits for adults for establishing overweight or (clinical) adiposity.

According to this nomenclature adiposity is divided into two different levels: adiposity level I (BMI = 30 - 34.9), adiposity level Il (BMI = 35 - 39.9), adiposity level III (BMI of more than 40) as well as morbid adiposity/super adiposity (BMI of more than 50). 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. At a BMI of more than 30 kg/ m² the mortality (death rate) increases by 1/3, at 40 kg/ m² by a factor of 3, at more than 40 kg/ m² by a factor 3 - 20 according the weight. For example, the risk of cancer is also increased (gynaecological carcinomas (endometrial carcinoma - 4 times; mamma- and cervix carcinoma - 2 times), prostate-, gall bladder-, colon- carcinoma)). However, 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. Also, the body's composition of the compartments - fat and fat-free mass (initially, body liquid is not considered here) may be included. According to this two-compartment model 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. Furthermore, 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. Also, the fat distribution is important for the risk assessment. An increased abdominal fat accumulation (central or android distribution type) poses a significantly higher risk than the peripheral or gyπoid distribution type. Therefore, the "waist-to-hip" ratio (the ratio of the circumference of the waist (cm) and the circumference of the hip (cm)) can also be included in the assessment. In women (> 0.8) or men (> 0.95) increased values are associated with an increased mortality.

Because 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. In childhood and adolescence 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. Because of the low incidence of adiposity-dependent diseases in childhood and adolescence and due to the lack of sufficient long term studies of the health risk of adiposity in childhood and adolescence, there are no established limits for the health risk of the body fat mass in this age range in contrast to the situation for adults. However, for the definition of overweight or adiposity in childhood and adolescence the BMI percentile can be used (obtained by extrapolation) that converges at the age of 18 to a BMI of 25 kg/ m² (overweight) or 30 kg/ m² (adiposity). For example, 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/ m² are the corresponding risk- related limits for adults. For a BMI of 25 kg/m² 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 comorbidities 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. Preferably, 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.

The development of 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/ m²) with an increased probability, in particular, an adiposity associated with co-morbidities.

For example, 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. For this purpose 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 Fn 14 or its natural ligand or parameters depending thereon, for example, in blood or in fat tissue.

The methods of treatment for adiposity known in the state of the art are essentially limited to a rigorous diet in combination with increased exercise. Presently, a known therapy is limited to the noradrenaline-serotonine-receptor-uptake-inhibitor "Sibutramin" (BASF, Ludwigshafen) or the lipase inhibitor "Xenical" (Roche, Basel, Switzerland). However, the application range of these medicaments is limited due to the occurrence of side effects/complications. For example, Sibutramin cannot be employed with high blood pressure - a typical complication for adiposity. Furthermore, the disease can only be treated symptomatically. One of the most frequent co-morbidities of adiposity is 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.

However, the administration of insulin to patients with type Il diabetes can result in hyperinsulinaemia (increased insulin level in the plasma, serum) due to the insulin resistance in the target tissues. Then, a therapy with insulin is no longer possible (Curr. Diab. Rep., October 3 (5), page 378 - 85, (2003)). In the context of a previously conducted project of the applicant for identifying protein factors having reievance for obesity, adiposity or type ii diabetes or cachexia, the applicant has demonstrated the existence of the protein TNFSF14, preferably in a trimeric configuration and in its soluble form as a new modulator of the differentiation of adipocytes.

This protein TNFSF14 unfolds therapeutic effects, preferably by directly affecting specific fat tissues within an individual or also indirectly, mediated by other fat tissue compartments or also by interacting with other tissues that are involved in the regulation of the energy homoeostasis.

It has further been demonstrated that this protein TNFSF14 plays an essential role for the treatment of adiposity/obesity. In particular, it was found that TNFSF14 is capable of inhibiting the differentiation or the function of adipocytes. This allows for the first time for a targeted treatment of adiposity/obesity.

The explanation for the found phenotype of the protein TNFSF14 results from the property of TNFSF14 as a receptor ligand that can elicit signal transduction upon binding:

The protein TNFSF14 is an integral class Il membrane protein of the tumor necrosis factor (TNF) ligand family (review in Cytokine & Growth Factor Reviews, 14, Granger and Ricert, p. 289 - 296 (2003)). It has a length of 240 amino acids and contains an N- terminal cytosolic domain, a type Il transmembrane domain as well as a C-terminal receptor binding domain. The soluble protein that is derived from TNFS F14 is 167 amino acids long and can activate the corresponding receptors. This receptor activation itself can cause changes in the cell amongst others, for example, cell survival or inflammatory processes or modulate them.

The protein TNFSF14 is a ligand for a member of the TNF-receptor superfamily that is also known as the lymphotoxin-beta-receptor (LT-β-R). Further receptors are the "herpes virus entry mediator" (HVEM) and DcR3. Until now a participation in immunological processes (T-cell proliferation, cytokine secretion, arteriosclerosis, "graft versus host disease") as well as an association with apoptotic processes has been shown for this ligand. The protein TNFSF14 stimulates the proliferation of T-cells and triggers the apoptosis of several tumor cells. The prevention of TNF-alpha-mediated apoptosis in hepatocytes is also known.

Surprisingly, the applicant was also able to demonstrate in the context of its work an inhibition of the differentiation of adipocytes or an inhibition of the lipid storage in adipocytes as a result of the TNFSF14-mediated activation of the LT-β-R signal transduction.

The LT-β-receptor is known as a receptor the TNF family ((Crowe et al., Science, 264, p. 707 - 710 (1994)). The LT-β-receptor binds heteromeric lymphotoxin complexes (LT-α/β) that contained LT-α subunits in combination with a further TNF-related polypeptide, the lymphotoxin-β (LT-β). These LT-α/β complexes are membrane- associated and for the most part have an LT-α1/β2 stochiometry (Browning et al., Cell, 72, p. 847 - 856 (1993), Browning et al., J. Immunol. 154, p. 33 - 46 (1995)). In analogy to TNF-receptors (TNF-R) and other TNF-like receptors it is assumed that the activation of the LT-β-R signal pathway occurs, when receptors are expressed in close vicinity on the cell surface (Crowe et al., Science, 264, p. 707 - 710 (1994)). This process is known as receptor cluster formation. The TNF- and LT-ligands are multivalent complexes that bind simultaneously to more than one receptor and can aggregate in this way. Receptor cluster formation has been extensively described as a means for receptor activation in other systems, in particular for the tyrosine kinase (Ullrich and Schlessinger, Cell, 61 , p. 203 - 212 (1990); Kolanus et al., Cell, 74, p. 171 - 183 (1993)). Therefore, the administration of LT-α1/β2 ligands and/or LT-β-R-activating agents can induce the cluster formation and, thereby, downstream signaling of LT-β-R molecules on the surface of target cells. The signal pathway of LT-β-R is known amongst others for activating reaction pathways like TNF-R that may lead to toxicity and cell death in tumor cells. This property of LT-β-R activation in tumor cells is also the basis for the use of LT-β-R- activating agents described in the patent application EP 809 510 B1 , including specific agonistic anti-LT-β-R antibodies for the treatment and reduction of neoplasia. However, whether such an activation may also have toxic effects on normal tissue and whether for this reason the receptor would provide a good tumor target molecule has not yet been elucidated in the state of the art.

Contrary to the effects of the LT-β-R activation described until now and their potential use in tumor therapy, the applicant has surprisingly found based on previous experiments that the LT-β-R signal pathway does not only play a role in the above described apoptotic and inflammatory processes, but has function in the differentiation of adipocytes, too. So it was demonstrated that ligands such as TNFSF14 but also LTa1 ^β2 and Lta2β1 that activate the receptor can inhibit the differentiation of fat cells. Because receptor activation can also be obtained by agonistic antibodies, for example, by those that recognize the corresponding LT-β-R, agonists, e.g. agonistic antibodies, can also be employed instead of natural ligands that specifically activate the above described LT-β-R in a targeted manner.

The functionality of the LT-β-R signal pathway for the differentiation of adipocytes that was surprisingly found by the experiments of the applicant provide the basis of an alternative treatment of adiposity/obesity and adiposity-associated diseases.

As already described above the treatment of adiposity/obesity and adiposity-associated diseases poses an enormous clinical problem. Previous therapeutic approaches have been and are still symptomatic, either by changing the diet, and in the case of adiposity- associated diabetes, for example, by the administration of PPAR-gamma agonists. 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. Moreover, the disadvantages of these symptomatic treatments (inter alia diabetes) are the occurrence of, for example, cardiovascular complications, diabetic nephropathies, neuropathies or retinophathies that need to be treated therapeutically, too. All previously known therapeutic approaches also have the momentous disadvantage that they are not capable of inhibiting the differentiation or the function of adipocytes.

Therefore, one object of the present application is to provide further means and routes for treating and/or preventing adiposity/obesity and, therefore, also to prevent adiposity/obesity-associated diabetes (type II). Summary of the invention

According to the invention the described problem is solved by providing further means and routes for the treatment and/or prevention of adiposity/obesity, by substances that effectively inhibit the differentiation and function of adipocytes. These substances are characterized according to the invention by their ability to induce LT-β-R-mediated signal transduction. Examples of such agents that induce LT-β-R-mediated signal transduction are the natural ligands or homologous ligands (or inter-species ligands) of LT-β-R. However, because a further problem with the therapeutic use of natural ligands of LT-β-R (such as, for example, TNFSF14) for treating adiposity/obesity and associated diseases is their sufficient production as well as their behaviour in the human body (protein stability, pharmacology, half life in serum, tissue distribution, undesired side reactions, etc.), agonists according to the invention, preferably antibodies that recognize and activate LT-β-R are used instead of natural ligands. Especially the use of antibodies that recognize and activate the receptor LT-β-R has the advantage in so far that these can be produced more easily and that they can be employed in patients in a more controlled manner due to their good pharmacological behaviour.

The described problem for providing agents for the treatment and/or prevention of adiposity/obesity is solved by the agonists according to the invention, such as antibodies and derivatives thereof, that recognize and activate the receptor LT-β-R. These agonistic antibodies and compositions containing these are employed for treating adiposity/obesity and further metabolic diseases that are associated with an increased or aberrant amount of fat cells by stimulating the LT-β-R signal pathway.

A preferred embodiment of the invention describes the use of at least one antibody directed against LT-β-R (anti-LT-β-R ab) for modulating the differentiation of fat cells, the use of a monoclonal or recombinant antibody (anti-LT-β-R mab) 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. A further embodiment of the invention describes binding molecules (for example, affilines, anticaiines, aptameres) that are directed against those epitopes of the receptor that cause activation of the receptor upon antibody binding.

Furthermore, the invention provides a new screening method for selecting LT-β-R- activating agents, wherein these agents comprise amongst others anti-LT-β antibodies, derivatives thereof, binding proteins and binding molecules and allow for the activation of the LT-β-R by binding at least one activating epitope of the LT-β-R.

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 LT-β-R in the modulation of the differentiation of fat cells is described by reference to examples and preferably comprises the following steps:

1) Undifferentiated human pre-adipocytes are seeded in cell culture dishes and are cultivated until post confluence.

2) These cells are incubated together with a specific adipocyte differentiation substance mixture which normally leads to a detectable differentiation into adipocytes with stored fat.

3) Simultaneously, the cells activated for differentiation in this way are incubated with the agents that are to be tested such as anti-LT-β-R antibodies, corresponding antibody preparations or formulations, or preferably with control antibodies.

4) The measurement of the differentiation of cells is done 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.

6) The strength of the differentiation-modulating activities of the tested agents that had been tested positive in the previous step is analyzed by serial dilution and subsequent determinations of dose-dependent changes in phenotype. This method can be modulated and adapted for the purpose of identifying new LT-β-R- activating agents, e.g. anti-LT-β-R antibodies, derivates thereof, binding proteins and binding molecules thereof.

For example, other cells or cell lines that express the LT-β-R naturally, in recombinant form as chimeric receptor or corresponding epitope-containing molecules can also be employed instead of differentiating pre-adipocytes for the described method for generating differentiation-modulating agents. When using said cells instead of pre- adipocytes the LT-β-R-activating effect of agents can be determined, for example, by detecting specific signal transduction events in the cell.

In this assay arrangement an anti-LT-β-R antibody (or a combination of antibodies) that inhibits or modulates the differentiation of adipocytes is an LT-β-R activating agent. These agents are used for the preparation of 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.

As mentioned above, the invention also relates to the use of functional variants of the above listed antibodies for the preparation of 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.

According to a preferred embodiment the adiposity/obesity is an uncomplicated adiposity. An uncomplicated adiposity is characterized in that there is no insulin resistance. Detailed description of the invention

The following description shall have the purpose of describing preferred embodiments of the invention in an exemplary manner.

Definitions

The term "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.

The term "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/obesity and/or high blood pressure and/or high cholesterol and/or insulin resistance.

The term "epitope" (or antibody binding site) is defined as the spatial chemical structure of an antigen that is recognized and bound by an antibody.

A monoclonal antibody (mab) recognizes a single epitope because all antibodies originate from one producing B-cell (= an antibody producing cell) and, therefore, have the same structure. 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. An epitope typically comprises several amino acids (for example, 5 - 7 amino acids) that can be located in linear sequence in the primary sequence of the antigen (= continuous epitope or linear epitope) or that can consist of different amino acids of the antigen that are not directly bond (= discontinuous epitope, non-linear epitope).

The term "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. The term "LT-β-R activating agent" relates to any agent that is capable of ligand binding to LT-β-R, cluster formation of LT-β-R on the cell surface or that enhances the LT-β-R signal pathway or that can influence how the cell interprets the LT-β-R signal on its inside. Examples of LT-β-R activating agents are TNFSF14, LT- α1/β2, soluble anti-LT- β-R antibodies, cross-linked anti-LT-β-R antibodies as well as multivalent anti-LT-β-R antibodies.

The term "LT-β-R signal pathway" relates to all molecular reactions in the context of the ligand-, antibody- or binding partner-mediated activation of LT-β-R and the resulting molecular reactions.

The term "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.

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 an antibody" according to the invention 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 or the differentiation of pre-adipocytes to mature adipocytes 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 LT-β-R signal transduction.

The term "anti-LT-β-receptor antibody" ("anti-LT-β-R ab") relates to all antibodies that recognize at least one epitope of the LT-β-receptor and bind to it.

The term "anti-LT-β-R antibody, poly- or monoclonal, cross-linking agent" relates to all agents that can bind to LT-β-R antibodies in solution either covalently or non- covalently, so that the antibodies bind to the surface of potential target cells and induce receptor signal transduction. 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 "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. Biotechnol., 14, pp.1116 - 1119, (1996); Klussmann et al., Nat. Biotechnol., 14, pp. 1112 - 1115, (1996)). Forms isolated in this way have the advantage that they are not degraded by naturally occurring ribonucleases and, therefore, have a greater stability.

Preparation of anti-LT-β-R antibodies for modulating the differentiation of adipocytes

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. For example, monoclonal antibodies can be prepared in accordance with the known methods of Winter & Milstein (Winter, G. & Milstein, C, Nature, 349, pp. 293 - 299, (1991)). Specific polyclonal antibody serums that are directed against the human LT-^β-receptor can be prepared by employing conventional methods by injecting animais, for example, goats, rabbits or mice, subcutaneously, for example, with an LT-β-receptor-derived protein or peptide or derivative that presents the activating epitope.

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.

For this purpose intraperitoneal or subcutaneous injections of additional agents that enhance the immune reaction (adjuvants, e.g. Freund's adjuvant) may also be employed. Polyclonal antiserums that contain the desired antibodies that are directed against the activating epitope of the LT-β-receptor can be expanded by repetitive intraperitoneal immunizations of mice with LT-β-receptor-derived protein or peptide or derivative in the absence of adjuvants. The immunization of animals with LT-β-receptors, derived proteins, peptides or derivatives thereof that represent the activating epitope can also be effected by either intraperitoneal or intravenous injections.

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 LT-β-receptor, 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 supernatants by means of protein A sepharose.

Various forms of anti-LT-β-receptor antibodies can also be prepared by employing standard methods for producing recombinant DNA (Winter and Milstein, Nature, 349, pp. 293 - 299 (1991)). For example, "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. According to the present invention the term 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 oiigospecific 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).

Furthermore, recombinant "humanized antibodies" that recognize the activating epitope of the LT-β-receptor 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 (interspecies) sequences in human antibodies and pose a lower risk for inducing an immune response in the treated individual.

The production of different classes of recombinant anti-Fn14 antibodies that recognize the activating epitope can also be achieved by preparing chimeric or humanized antibodies with anti-LT-β-R variable domains and human constant domains (CH1 , CH2, CH3) that had been isolated from different classes of immunoglobulins. For example, anti-LT-β-R 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)).

As an alternative to classical antibodies it is also possible to employ so-called "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)). For other protein scaffolds it is known that they are alternatives for antibodies (Skerra, J. MoL Recognit, 13, pp. 167 - 287, (2000)). All these protein-derived alternatives for antibodies can be denoted binding proteins. For this reason and according to the present invention the term 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 LT-β-receptor, proteins derived from these, peptides or derivatives thereof, that were produced recombinant^, and if required, were modified.

Identification of epitopes of LT-β-R that result in receptor activation upon binding the anti-LT-β-R antibody

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 LT-β-R, can result in its activation. For doing so the mere binding of the antibody is necessary but not sufficient. Moreover, 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.

The position of such an epitope in the antigen can be mapped, if at least one activating antibody (also called an agonistic antibody) is available. For this purpose the protein of the receptor, i.e. LT-β-R, is partitioned into distinct portions, what may either be done by the recombinant production of partial sequences of the receptor but also by synthesizing partial peptides of the receptor. Such fragments or partial peptides of the receptor are then analyzed for the ability to be recognized by agonistic antibodies. This analysis may be done by established techniques, e.g. ELISA₁ or by more recent binding techniques for antibodies to peptide arrays. For practicing the latter overlapping partial peptides of the receptor LT-β-R are immobilized in the form of arrays. These arrays are supplied with an agonistic anti-LT-β-R antibody after pre-treatment with blocking buffer and are incubated for a sufficient time that depends on the antibody and washed afterwards. Finally, the agonistic antibody bound to specific peptides is detected on microarray scanners. The binding to partially overlapping peptides that are detectable in this way that are derived from the LT-β-R allows for the accurate mapping and, thereby, definition of the epitopes, that lead to the induction of the receptor signal transduction upon antibody binding. These peptides can then be used for the targeted preparation of further agonistic anti-LT- β-R antibodies that are all characterized in that they recognize the defined peptide. By combinations of and combining several peptides it is also possible to generate agonistic anti-LT-β-R antibodies that are directed against discontinuous epitopes, i.e. those antibodies that simultaneously recognize two or more partial sequences that are not connected to each other in the primary sequence. It is also possible to use artificial chemical structures as epitopes that correspond to the described natural continuous or discontinuous epitopes in shape, charge, structure and other relevant properties for the epitope binding without the original peptide sequence being contained in these structures. These peptides, epitopes or artificial chemical structures that correspond functionally to the epitopes may be used for immunizing animals for generating, for example, polyclonal or monoclonal antibodies, as well as for generating antibody derivatives and binding proteins by recombinant technologies. In addition, it is also possible to use these activating epitopes as immunogens for preparing activating antibodies directly in humans. This allows for the vaccination of patients, whereby their adiposity and their follow up damages may be treated.

The identification (by means of the above methods) of epitopes that lead to receptor activation upon binding serves amongst others for generating agonistic antibodies with the assistance of rational targeted methods. On the other side, it is also possible to gain information on epitopes that lead to receptor activation upon binding, and, in particular about antibodies, that bind these epitopes, by competition experiments. In this manner already available agonistic antibodies, for example, those that are presented in Example 2 and Figure 6 as agonistic anti-LT-β-R antibodies, can be used for identifying and characterising further agonistic antibodies. Such an antibody is, for example, the agonistic, human-specific polyclonal antibody (goat IgG anti-human lymphotoxin-beta- receptor polyclonal antibody, AF629, Fig. 6). Because the antibodies described in Example 2 and Figure 6 as agonistic anti-LT-β-R antibodies recognize an epitope of the LT-β-R that imparts receptor activation upon binding, the antibodies or antibody preparations or formulations that bind to the same epitope will activate the receptor, too. Competition experiments, wherein the antibody to be analyzed is tested, whether it can prevent the binding of a given (in this case, for example, the antibody described as agonistic anti-LT-β-R antibody in Example 2 and Figure 6) antibody to the receptor (= competes) when provided in excess can be used for finding antibodies that bind to the same epitope that is bound by the used agonistic anti-LT-β-R antibody. Said competition analysis that may be performed by a skilled person with standard methods may be used accordingly for the analysis of the potential binding of new antibodies to an activating epitope of LT-β-R.

Modulation of the differentiation of fat cells by specific activation of the LT-β-R- mediated signal transduction

According to a preferred embodiment the anti-LT-β-R antibodies, antibody derivatives or binding proteins thereof used according to the present invention function by inhibiting the formation of terminally differentiated adipocytes. For this purpose the formation of terminally differentiated adipocytes may be inhibited in that the differentiation to terminally differentiated adipocytes is inhibited, in particular by inhibiting the transcription signal pathway for differentiation. On the other side, the formation of terminally differentiated adipocytes can be inhibited by inhibiting lipogenesis or assisting lipolysis. The inhibition of the differentiation to terminally differentiated adipocytes can be done by measuring the lipids (e.g. by staining with Nile Red). Reporter gene assays such as, e.g. for PPAR-Y or CEBP-β or -δ, allow for measuring the inhibition of the transcription signal pathway. By staining with 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 the LT-β-R is described for the natural receptor ligands TNFSF14, LT-α1/β2 as well as for activating anti-LT-β-R antibodies in Example 2. In murine cells as well as in freshly prepared human pre-adipocytes the effect of the ligand- (TNFSF14) and antibody- (anti-LT-β-R) mediated signal transduction may be illustrated in a dose-dependent manner (see Figures 1 - 3, 5, 6). Because an involvement of the LT-β-R-mediated signai transduction in apoptotic processes and cell death (for example, in tumor cells) is already known, it was also analyzed whether the inhibition of the differentiation of adipocytes is associated with the induction of apoptosis or cell death. For example, the identification of apoptosis is possible with the CDD⁺ assay (cell death detection ELISA PLUS, Roche Diagnostics GmbH, Mannheim, Germany), DiOC6 assay, annexin V assay or CaspaTag™ assay (CaspaTag™ caspase-3 (DEVD) activity kit, lntergen Company, Purchase, NY₁ USA). For example, the determination of the live cell count is possible with the "Alamar Blue" or "CellTiterGlow" assays.

The possibility of toxicity or the induction of apoptosis by activating the LT-β-R was assayed by the applicant in murine and human cells and is described in Example 3. It was able to demonstrate that for the used conditions there was no toxicity. In these experiments it was also demonstrated that the inhibiting effect is reversible. This was also demonstrated by removing the protein, which subsequently allows a new entry of the cells into the differentiation process. Obviously, this is only possible for living cells, a fact that was also confirmed by microscopic measurements of the living cells.

Therapeutic use of antibodies and other activators of the LT-β-R-mediated signal transduction for treatment of adiposity/obesity and associated diseases

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 LT-β-R antibodies is administered to the patient. Preferably, the patient suffers from adiposity/ obesity. For this method according to the invention all of the embodiments illustrated above for the use according to the invention apply, too.

As already described above, next to the inhibition of the differentiation of pre-adipocytes in adipocytes the factors TNFSF14 and the anti-LT-β-R antibodies found according to the invention allow for the inhibition of the function of mature or maturing adipocytes, for example, by inhibiting lipogenesis.

The observations that the targeted receptor activation of LT-β-R by recombinant supplementation of the receptor-activating ligand TNFSF14 can reduce the weight of animals (see Example 4, Figure 7) is proof for the therapeutic concept described herein. Additional proof comes from animal studies where monoclonal agonistic anti-LT-β-R antibodies were also able to reduce the weight in animals (see Example 5, Tables 1 and 2) both on a normal and on a high caloric diet.

According to the invention, by inhibiting the complete or partial suppression of a biological function is understood. In this specific case the inhibiting function is the differentiation of adipocytes in patients with adiposity/obesity. According to the present invention the term "inhibitor" describes an agent, such as an anti-LT-β- antibody, that preferably inhibits the process of fat storage by itself. In this case the activating antibody can be denoted an inhibitor. The inhibition can, for example, be achieved by the induction of the LT-β-dependent signal transduction and subsequent modulation of the expression of regulated specific genes.

According to a preferred embodiment the inhibitor used, e.g. the anti-LT-β-R antibody, elicits its effects by inhibiting the formation of terminally differentiated adipocytes. Preferably and in an exemplary manner, this can take place by interfering with the differentiation to terminally differentiated adipocytes, in particular, by modulating the LT- β-R transcription signal pathway for the differentiation of pre-adipocytes.

Furthermore, 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:

For the preparation of the pharmaceutical composition according to the invention 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, ε-aminocaproic acid or pepstatin A or chelators, such as EDTA₁ gel formulations, such as white vaseline, paraffin with low viscosity or yellow wax, depending on the mode of the administration.

Further suitable additives are, for example, detergents, such as, for example, Triton X-100 or sodium deoxycholate, but also 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 or a protein, such as, for example, bovine or human serum albumin. Detergents, polyols and/or amphoteric substances are preferred.

Preferably, 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/liter, 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). The choice of the adequate buffer generally depends on the desired buffer molarity. For example, a phosphate buffer is suitable for injection or infusion solutions.

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. In principal, 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. For example, 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) can further increase this effect.

According to a preferred embodiment 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 listing of the above embodiments is intended to describe the present invention more clearly, but not to limit it.

Furthermore, the invention relates to a method for the treatment of a patient, wherein an effective amount of one or more of the above-defined inhibitors is administered to the patient. Preferably, the patient suffers from adiposity/obesity or diabetes type Il or from diabetes type ll-related diseases.

Examples In the following the invention will be illustrated by examples and figures that are by no means intended to iimit the subject-matter of the invention but instead represent embodiments.

In particular, it is shown in the examples that 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.

Furthermore, it is demonstrated that the receptor LT-β-R, 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.

Moreover, it is demonstrated that the activation of the receptor LT-β-R that effects the modulation of the differentiation of fat cells leads to no toxic effects in the system of murine and human cell cultures that is used in the present case.

In addition, it is shown that expressed, soluble, murine, homologous variants of TNFSF14 or their protein fragments are capable of reducing the gain in weight in mice without causing general toxic effects.

It was also shown that antibodies that cause the fat cell-modulating phenotype by receptor activation bind to defined epitopes of the receptor LT-β-R and, thereby, initiate signal transduction. This fact is an essential pre-requisite for the rational generation of defined antibodies that activate the signal transduction by the targeted binding to these epitopes of the receptor.

In addition, it was shown that monoclonal agonistic anti-LT-β-R antibodies are capable of reducing the gain in weight in mice without causing general toxic effects. This was validated for animals on a normal and on a high caloric diet.

Description of the figures: 4 .

Fig-

This illustrates a dose/response curve that shows the lipid content of 3T3-L1 cells in dependency of the added amounts or a recombinant soluble or secreted or soluble protein fragment TNFSF14. The measured results are provided as relative fluorescence units (RFU) on day 5 after the addition of the protein.

Fig. 2:

This illustrates the percentage inhibition of the lipid storage on day 8 in the form of a dose/response curve by recombinant secreted or soluble protein fragments of TNFSF14 in primary human adipocyte cell cultures of subcutaneous origin. For this purpose 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 means a low lipid storage in the differentiating human adipocytes, a strong signal means a strong lipid storage.

Fig. 3:

This illustrates the inhibition of lipid storage by added recombinant soluble protein fragments of alpha 1/beta2-lymphotoxin, alpha2/beta1-lymphotoxin in comparison to TNFSF14 in primary human adipocytes of subcutaneous origin. The measured results are specified as relative fluorescence units (RFU) on day 8 after the first addition of the respective protein. A low signal means a low lipid storage in the differentiating human adipocytes, a strong signal means a strong lipid storage

Fig. 4: Receptor identification mHVEM-mLT-β-R

This illustrates the identification of receptors of TNFSF14 ((A) HVEM-receptor and (B) lymphotoxin-β-receptor) by specific receptor-antibodies (goat IgG anti-mouse HVEM polyclonal ab, R&D systems, Wiesbaden/Germany, cat.no. AF2516 and rat lgG2a anti- mouse-lymphotoxin-beta receptor monoclonal antibody, clone 4H8WH2; lmmunogen amino acids 31 - 221 , this corresponds to the complete extracellular domain of the mature mouse receptor protein; Fa. Axxora, Germany Cat. No. ALX ALX-804-145) and a suitable fluorophore-labelled secondary antibody and the fluorescence-activated cell sorting method (FACS) on 3T3-L1 cells. The measured results are specified as fluorescence intensities (FL-1) relative to the event number. The fluorescence intensity profile is compared in the absence (D) or presence (■) of the specific receptor antibody. A shift in the fluorescence intensity to stronger intensities (shift to the right) means a binding of the specific receptor antibody together with the fluorophore-labelled secondary antibody to the cell surface. This illustrates the receptor exposition on the cell surface.

Fig. 5:

This illustrates the dose/response curves of an agonistically acting antibody for the lymphotoxin-β-receptor (rat lgG2a anti-mouse-lymphotoxin-beta receptor antibody, clone 4H8WH2; Immunogen, amino acids 31 - 221, this corresponds to the complete extracellular domain of the mature mouse receptor protein; Axxora company, Germany, cat. no. ALX ALX-804-145), and of TNFSF14 as well as to further known alternative ligands for the lymphotoxin-β-receptor - lymphotoxin a1b2 and lymphotoxin a2b1. The lipid content of 3T3-L1 cells was measured dependent on the added amount of a recombinant secreted or soluble protein fragment TNFSF14, alpha1/beta2-lymphotoxin, alpha2/beta1 -lymphotoxin or the mouse-specific monoclonal antibody, respectively. The measured results are specified as relative fluorescence units (RFU) on day 5 after the addition of the respective protein. A low signal means a low lipid storage in the differentiating human adipocytes, a strong signal means a strong lipid storage.

Fig. 6:

This illustrates the concentration-dependent inhibition of lipid storage by added antibodies (goat IgG anti-human lymphotoxin-beta receptor polyclonal antibody, Immunogen: extracellular domain of the human lymphotoxin-beta receptor; Fa. R&D Systems, Germany, Cat. No. AF629) in comparison to TNFSF14 in primary human adipocytes of subcutaneous origin. Also shown is the absence of an inhibition of lipid storage when an HVEM- specific antibody (rat lgG2a anti-human HVEM monoclonal antibody, clone Laury-1 , Immunogen: extracellular domain of HVEM, amino acids 39- 200, Fa. Axxora, Grϋnberg Germany Cat. No.: ALX-804-814) is employed. The measured results are specified in percent versus lipid storage of normal differentiated cells. The data was taken on day 8 after the first addition of the respective protein and the induction of differentiation. A low signal means a low lipid storage in the differentiating human adipocytes, a strong signal means a strong lipid storage.

Fig. 7:

This illustrates the restriction of the gain in body weight in male C57BL/8 mice two weeks after injection of a suitable expression vector in dependency on the extent of the expression of the soluble or secreted protein fragment of murine TNFSF14. The extent of the expression of the protein fragments was determined by an ELISA of serum samples. Also, the negative correlation of protein expression and gain in body weight is shown.

Fig. 8:

This illustrates the insulin-dependent glucose uptake in primary human adipocytes of subcutaneous origin. Ineffective (10^"1° M) and effective (1fJ⁷ M) concentrations of insulin were employed. Effective insulin concentrations result in an increased cellular glucose uptake under the selected conditions (control). The effects of the co-incubation of added protein TNFSF14 (100 ng/ml) are also shown. TNF-alpha (50 ng/ml) served in this context as a positive control, because its interference with the insulin-dependent glucose uptake in adipocytes has already been described. The measured results are specified as "insulin-dependent uptake of pmol glucose within 20 min".

Fig. 9:

This illustrates assays for the proliferative activity or the cytotoxicity of TNFSF14 on subconfluent 3T3-L1 cell cultures. The proliferating activity or the cell count was determined by determining the living cell number by the AlamarBlue assay and the measured results are specified as relative fluorescence units (RFU).

Table 1 :

This illustrates the significant effect of an agonistic monoclonal anti-LT-β-R antibody on adipose tissue weights in mice fed a normal chow diet compared to a control group. Data shown are mean ± standard deviation (n = 10). Results are considered significant if p< 0.05. Young male C57BI/6 mice, fed a normal mouse chow diet, were injected with an lgG2a directed against LT-β-R (50 μg/mouse i.p. three times weekly). Control mice were similarly injected with control isotype lgG2a. Study period was six weeks. At autopsy, the weight of the epididymal fat pads and of the subcutaneous dorsal fat was significantly smaller in the mice treated with specific antibody. Treatment with an antibody against the LTB-R thus had significant effects on adipose tissue weights.

Table 2:

This illustrates the significant effect of an agonistic monoclonal anti-LT-β-R antibody on adipose tissue weights in mice on a high caloric diet compared to a control group. Results are considered significant if p< 0.05. Young male C57BI/6 mice, fed a high fat- diet for twelve weeks prior to treatment, were injected with an lgG2a directed against LT-β-R (Group A; 50 μg/mouse i.p. three times weekly). Control mice (group B) were similarly injected with control isotype lgG2a. Study period was six weeks. At autopsy, (abdominal and subcutaneous) adipose tissue weighed significantly less in the treated group, while the weight of other tissues did not differ. Thus, in male mice fed a high-fat diet for twelve weeks, treatment with an antibody against LTB-R resulted in significant weight loss of adipose tissue in the treated group.

Example 1:

Cultivation of pre-adipocyte cells, their induction for differentiation and the measurement of intracellular stored lipids

For the propagation and analysis of murine 3T3-L1 cells:

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 originated 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 10⁵ cells/T-75 flask. There is no medium exchange until the next passage. The seeding of the 3T3-L1 cells for the experiment was conducted in 96-well microtiter plates: The 3T3-L1 cells were seeded at a cell density of 1.5 x 10⁴ cells/well in 200 μl 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 (GM) had the following composition: DMEM, 10% FCS, 1% penicillin-streptomycin, 2% glutamine, 1% Na-pyruvate). The differentiation medium (DM) 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 μM dexamethasone).

For the isolation of adipocytes from fat tissue and the cultivation of primary human 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 ⁰C 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 NH₄CI₁ 5.7 mM K₂HPO₄, 0.1 mM EDTA, pH 7.3). After a maximum incubation of 10 min at room temperature it was filtered over a 150 μM filter and centrifuged as described above. Because the remaining pellet still contained remaining connective tissue, it was again purified with a 70 μM filter after being taken up in pre-adipocyte medium (DMEM/F12 (Invitrogen GmbH, Karlsruhe) supplemented with 8 mg/l biotin, 4 mg/l pantothenate, 1.79 g/l NaHCO₃ and 55 mg/l pyruvate). The cells were counted, centrifuged again and resuspended in DMEM/F12 with 10 % FCS and 50 μg/ml gentamycin. The seeding was done at a density of 40000 to 55000 cells/cm². The cells were incubated at 37 °C and 5 % CO₂.

Induction for differentiation and addition of the supernatant or the protein to be tested onto 3T3-L1 cells:

Three days after seeding the 3T3-L1 cells 140 μl medium of the 3T3-L1 cells were taken and 50 μl growth medium was added, wherein suitable amounts of recombinant protein (0 - 3 μg/ml protein) or antibody (for example, 0 - 10 μg/ml) had been dissolved. In addition, 100 μl 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.

Differentiation of human pre-adipocytes and SGBS cells:

Human pre-adipocytes were seeded at a density of 40000 to 55000 cells/cm². For SGBS cells (described in Wabitsch et al., 2001 , Int. J. Obes. Relat. Metab. Disord. 25:8 - 15) the cell density was lowered by a factor of 10. After the seeding the cells of the primary culture remained for 20 h, the SGBS cells remained for 3 days in proliferation medium (pre-adipocyte medium with 10 % FCS and 50 μg/ml gentamycin). For inducing the differentiation the cells were washed twice with PBS and then incubated in serum-free differentiation medium (pre-adipocyte medium with 66 nM insulin, 1 nM T₃, 100 nM hydrocortisone, 10 μg/ml transferrin, 50 μg/ml gentamycin). Also, for the first 3 days 1 μg/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:

For quantitatively determining the storage of cellular lipids the Nile Red reagent (Molecular Probes, Leiden, Netherlands; CAS number 7385-67-3) (Nile Red staining solution: 4 μg/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. For performing the Nile Red assay 140 μl medium of the 3T3-L1 cell cultures were removed and 200 μl PBS were added on day 5 (3T3-L1 cells) or days 8 - 12 (primary human pre-adipocytes cultures) after induction for differentiation. Then, 150 μl liquid were removed and 50 μl Nile Red staining solution were added. The incubation of the plates was done for 4 hours at 37 ⁰C in a CO₂ incubator. The read out was done in a fluorescence reader at an excitation of 485 nm and an emission of 590 nm (200 msec readout time). The measured results are specified as relative fluorescence units (RFU) or as percentages relative to normal differentiated (non-inhibited) controls. Example 2:

Inhibition of the lipid storage in cell cultures by activating the receptor with receptor-specific antibodies or natural ligands.

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.

At first, a receptor was activated by its natural ligand. In the present case 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 TNFSF14 (R&D Systems GmbH, Wiesbaden: Cat No.: 664-LI [CD33 signal (met 1-17)/10 x His/GGGSGGGSGGGSIEGR/]-Asp-74-Val-240). The results demonstrate that the lipid storage of murine 3T3-L1 cells cultures (Fig. 1) as well as in primary human adipocytes (Fig. 2) can be inhibited during differentiation by administering the recombinant soluble protein fragment of TNFSF14 in a dose-dependent manner. 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. In both cellular systems there was a half maximum effective concentration of about 2 ng/ml for the recombinant soluble protein fragment of TNFSF14. A comparable inhibition of the differentiation was also accomplished for primary fat cells of omental origin and the human adipocyte cell line SGBS. The alternative ligands human lymphotoxin alpha1/beta2 and lymphotoxin alpha2/beta1 (100 ng/ml each) for the lymphotoxin-β-receptor displayed an inhibition of the storage of lipids in 3T3-L1 cells (Fig. 5) or in primary human adipocytes (Fig. 3). On murine 3T3-L1 cells the agonistic mouse-specific monoclonal antibody (rat lgG2a anti-mouse-lymphotoxin-beta receptor monoclonal antibody, clone 4H8WH2, Fig.5) directed against the lymphotoxin-β-receptor (LT-β-R) of TNFSF14 was employed. The agonistic human-specific polyclonal antibody (goat IgG anti-human lymphotoxin-beta- receptor polyclonal antibody, AF629, Fig. 6) was used for human adipocytes. The results demonstrate that the lipid storage in murine 3T3-L1 cells as well as in primary human adipocytes (Fig. 6) during differentiation can be inhibited in a dose-dependent manner by the addition of an agonistic antibody (4H8WH2 or AF629). 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. The addition of 1 μg/ml human-specific receptor antibody led to an inhibition of differentiation that is comparable to the employment of 100 ng/ml natural ligand (TNFSF14) (Fig. 6). The addition of 50 ng/ml mouse-specific receptor antibody led to an inhibition of differentiation in 3T3-L1 cell cultures that was comparable to the use of 15 ng/ml natural ligand (TNFSF14) (Fig. 6). The use an antibody directed against the second cell-bound receptor of TNFSF14, HVEM, showed no inhibition of adipocyte differentiation (Fig. 6). However, the antibody had not been described as agonistic and such an activity was also not established in the present case. However, an activation of the lymphotoxin-β-receptor in 3T3-L1 cells alone seems to be sufficient to realize an inhibition of differentiation that is similar to that of the natural ligand as it is shown in Fig. 5.

Even though the presence of the receptors had already been demonstrated by functional data, the following receptors were also identified in direct detection experiments: Figure 4 shows the analysis of the expression of the receptor as a target for the activating antibody in mouse 3T3-L1 cell cultures. The mouse HVEM receptor of TNFSF14 was detected with the antibody pair of receptor-specific rat IgG antibody (company Alexis Biochemicals, catalogue number ALX-804-156) and the secondary Alexa-Fluor goat-anti- rat IgM-specific antibody A-21212 of the company Molecular Probes. The lymphotoxin-β receptor of TNFSF14 was confirmed with the antibody pair of receptor-specific rat IgG antibody (company Alexis Biochemicals, catalogue number ALX-804-145) and the secondary Alexa-Fluor goat-anti-rat IgG-specific antibody (company Molecular Probes, catalogue number A-11006). The staining of subconfluent 3T3 cells was effected according to following scheme: seeding of 200000 cells in a 6 well dish. 24 h later staining and measuring in the FACS device: For doing this the cells were trypsinised and pelleted and subsequently incubated for 1 h on ice in 200 μl buffer solution (PBS + 1 % FCS) or in buffer solution with receptor-specific antibody according to the manufacturer^'s instructions (1 - 5 μg/ml). Thereafter, 1 ml PBS + 1% FCS was added and the cells were pelleted. The pellets were taken up in 200 μl PBS buffer with 1% FCS in which the secondary antibody (2 μg/ml) had been dissolved. Further incubation on ice for another 45 min followed. Subsequently, the cells were placed in 1 ml PBS + 1% FCS and pelleted. After again being taking up in 500 μl buffer the cells were subsequently taken to the FACS device for measurement. The measurement of the fluorescence intensities was only conducted for living cells.

The result shows that the lymphotoxin-β receptor for TNFSF14 was detected on murine 3T3-L1 cells. Surprisingly, the HVEM receptor for TNFSF14 was detected, too, that had mostly been described as being limited to lymphocyte cells.

Example 3:

Investigation of possible adverse effects after receptor activation

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.

Specifically the determination or the exclusion of cytotoxic mechanisms during the observed inhibition of adipocyte differentiation was assayed as follows: 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 TNFSF14 was done. In the course a dose- response relationship was investigated over a concentration range of 0.1 to 500 ng/ml protein. 72 h after the addition of the proteins in medium with 2 % FCS 10 μl AlamarBlue reagent (BioSource company) were added to 100 μl culture medium and incubated for 4 h at 37 ⁰C. The AlamarBIue reagent provides a substrate for living cells that is reacted enzymatically and the product can be measured fluorometrically at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. By doing so an indirect cell count determination is provided in the linear measurement range. These results demonstrate that subconfluent 3T3-L1 cell cultures were neither activated for an increased proliferation by the addition of recombinant soluble protein fragment of TNFSF14 (Fig. 9) nor was a cell death detectable. These results were also confirmed for differentiating 3T3-L1 cell cultures.

For determining the acute in vivo toxicity of a receptor activation the following can be assayed: 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"). 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. In a dose tolerance test 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. In particular, it was shown herein that the one-time intravenous administration of up to 10 mg/kg body weight in the case of recombinant soluble protein fragment of TNFSF14 into female C57/BI6/J mice led to no clinical symptoms within 48 that were caused by the test substance.

Because the differentiation of adipocytes is inter alia an insulin-dependent process, it was to be determined, whether the receptor activation can lead to a negative modulation of the insulin signalling in adipocytes. In these experiments that are shown in Figure 8 assays relating to the interference of TNFSF14 with the insulin-dependent glucose uptake in 3T3-L1 cells were conducted. 500000 primary adipocytes on day 12 after the induction of differentiation were kept in a 3 cm dish and treated as follows for determining the insulin-dependent glucose uptake: Experimental set up: Controls: 1 cell culture for background (O' insulin stimulation). An empty well/dish for determining the background. The stimulation was done with 10^"7, without or 10^"10 insulin, because 10^'10 insulin is not stimulating in this context. In each 3 cm dish there were 800 μl medium DMEM/Ham F12 with 5 mM glucose. Differentiated cells were washed four times with 2 ml warm PBS. Then 800 μl 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 ⁰C. Subsequent addition of insulin. Plates were incubated for 15 min at 37 ⁰C at the lowest agitation frequency. 2-Deoxy-D-[1-H³]- 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-H³]-glucose. After 15 min incubation 8 μl of the diluted 2-deoxy-D-[1 -H³]-glucose were added. The plates were again incubated with light agitation for 20 min at 37⁰C. The plates were placed on ice after 20 min. The medium was suctioned off by a pump. Cells were washed 3 times with 1 ml ice cold PBS each. 500 μl IGEPAL (Sigma I 3021) were added per well followed by 20 min lysis on ice. The complete volume of each well was pipetted into the scintillation cups. In addition, 500 μl IGEPAL plus 8 μl of the diluted 2- deoxy-D-[1-H³]-glucose into one cup. In each cup 3.5 ml of the scintillation liquid (Rotiszint eco plus, Roth, 0016.2) were pipetted. Measurement in the scintillographic device over one minute per sample followed. The readout was done with the help of the program Mikro Beta Windows. For the presentation of the results the basal glucose uptake is subtracted, so that only the insulin-dependent part of the glucose uptake is shown.

The receptor activation by TNFSF12 in this experimental set up demonstrated no negative influence of the insulin-dependent glucose uptake.

Example 4:

Reduction of the weight or the gain of weight in the mouse model

Preferred animal models are those that correspond to the human situation of adiposity/obesity or type Il diabetes in a specific manner. Preferably, 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.

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").

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. In a dose tolerance test 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. For further experiments the above-determined maximum dose is reduced to the lowest dose that still has a therapeutic effect. In the following test for determining the change in weight, said determined dose is administered parenterally (intravenously, intraperitoneally, subcutaneously or intramuscularly). The intraperitoneal, intravenous or subcutaneous application is preferred.

For determining the reduction of the gain in weight in normal weight mice 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. During and after these experiments the gain in weight is determined by weighing and/or determining the BMI.

For treated mice that are kept on a normal calorie diet a reduction in weight or reduced gain in weight is observed contrary to untreated mice. For treated mice that are kept on a high calorie diet a lesser gain in weight or a weight reduction is observed in comparison to untreated mice. This reduction in weight can also be attributed to a reduced fat tissue mass.

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.

For treated mice that were kept on a normal calorie diet or continued high calorie diet a stronger reduction in weight was shown in comparison to untreated mice. For untreated mice on a high calorie diet a further increase in weight is observed. However, for treated mice a weight reduction or a lesser gain in weight is noted.

In an animal experiment the following was demonstrated: 8 five week old male C57BL6/J mice were each injected with an expression construct for recombinant secreted and soluble murine protein fragment of TNFSF14. 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.

A TNFSF14 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 TNFSF14.

The expression constructs were prepared by performing an RT-PCR with a suitable murine RNA source (mouse thymus RNA) and with suitable primers (comprising the sequences coding for: mouse-TNFSF14: Asp-72 - Val-239). In addition, 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.

Used primers: m14-F-His:

5^'-CATCACCATCACCATCACGATGGAGGCAAAGGCTCCTG- 3^' (compare to SEQ ID

NO: 2) m14-R:

5^'-AGATCTCGAGTCATCAGACCATGAAAGCTCC- 3^' (compare to SEQ ID NO: 3)

The reaction product was inserted directly into the vector pSECTag/FRTΛ/5-His-TOPO (Invitrogen, Karlsruhe, Germany) according to the instructions of the manufacturer for the cloning of PCR products. In this way the secretion signal of the protein for the Ig-kappa chain precedes the protein fragment of TNFSF14 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:

Mouse-TNFSF14-reading frame (compare to SEQ ID NO: 1):

METDTLLLWVLLLVWPGSTGDAAQPARRARRTKLALHHHHHHDGGKGSWEKLIQDQR

SHQANPAAHLTGANASLIGIGGPLLWETRLGLAFLRGLTYHDGALVTMEPGYYYVYSKV

QLSGVGCPQGLANGLPITHGLYKRTSRYPKELELLVSRRSPCGRANSSRVWWDSSFL

GGWHLEAGEEVWRVPGNRLVRPRDGTRSYFGAFMV Subsequently, 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/FRTΛ/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 pXAP02 (mTNFSF14).

The transfection of the plasmids in C57BL6/J mice coding for recombinant secreted and soluble murine protein fragment of TNFSF14 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. The method in brief: 50 μg endotoxin-free prepared plasmid were administered in PBS solution in an amount of solution corresponding to 10 % of the body weight of the mouse to be injected with a velocity 1 ml/5 sec. Then, the animals were weighed up to 8 weeks after the injection on a weekly basis. The content of the expressed secreted soluble murine protein fragment of TNFSF14 was determined by an ELISA in obtained blood sera. ELISA antibody pairs for murine TNFSF14: For coating: anti-penta-HIS antibody (Qiagen, cat. no. #34660) and for the specific identification: biotin-conjugated anti-hLIGHT antibody (R&D Systems, cat. no. # BAF664; that had shown a cross reactivity against mouse TNFSF14 in preliminary tests).

The experiments described in Fig. 7 have demonstrated that the degree of expression of murine, recombinant, secreted, soluble protein fragment of TNFSF14 clearly correlates negatively with the observed gain in weight during the period of investigation. Based on the present data a correlation coefficient of -0.71 resulted for TNFSF14 (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.

The trend indicated by the correlation was also observed by weighing selected fat tissue deposits (,,epididymal 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 corresponds to the reduction of the weight of selected fat deposits.

More detailed experiments are preferred, wherein at the time point of administering the inhibitor 12 week old C57BI6 mice were kept on a normal calorie diet or a high calorie diet and subsequently on a high calorie diet for another 8 weeks until the time of injection. Under these conditions of a diet-induced adiposity/obesity it is to be expected that the presence of the protein fragments of TNFSF14 or an activating anti-LT-β-R ab described herein will lead to a significantly reduced gain in weight or an absolute reduction in weight that is preferably caused by a significantly reduced weight of the fat tissue deposits.

Example 5:

Reduction of the weight or the gain of weight in the mouse model using monoclonal anti-LT-β-R antibodies

As noted in example 4 above, the preferred animal models are those that mimic the situation in human adiposity/obesity or type Il diabetes. Accordingly, a particularly preferred embodiment is the use of anti-LT-p-R antibodies or derivates thereof to reduce the weight or weight gain in animals, preferably rodents such as, for example, mice or rats, as well as dogs, pigs and primates. The adiposity can be diet-induced (DIO), spontaneous, genetically predisposed, and optionally associated with an insulin resistance or type Il diabetes.

In an animal experiment the following was performed: a treatment group and a control group, each comprised of 10 male C57BL6/J mice were tested. The treatment group was injected a rat-anti-mouse LT-β-R lgG2a (Alexis, antibody 4H8 WH2, code ALX-804-145, lot L15482) (treatment group), the control group was injected a corresponding isotype control antibody (rat lgG2a from R&D Systems). Both antibody preparations were diluted to 0.5 mg/ml with saline, and stored at 4°C. Mice were injected, three times weekly (Monday, Wednesday and Friday), in the morning, with 0.1 ml_ (50 μg) of a preparation intraperitoneally. All animals were housed during the experiment in clean-conventional animal rooms (relative humidity 50-60%, temperature -21 °C, light cycle 6 am to 6 pm). The mice were fed a normal chow diet (normal rodent chow; Ssniff, Bioservices, Schaijk, the Netherlands)kept. Food and acidified water were given ad libitum. Mice were housed groupwise (δ per cage) in macroion cages with bedding of wood chips.

In another experiment, a treatment group and a control group were treated and kept as described above with the following modifications. Instead of a normal chow diet the animals were fed a high-fat diet containing 24% (w/w) lard (Hope Farms, Woerden, the Netherlands) for twelve weeks before treatment and feeding on the high-caloric diet was continued throughout the study.

The study period for each study was six weeks. At the end of the study period, mice were prepared for autopsy. After a 4-hr fast, mice were weighed, and then anaesthetized with FFM (fentanyl citrate, fluanisone, midazolam). The following tissues were weighed: heart, lungs, liver, kidneys, spleen, epididymal fat pads, perirenal fat and intestinal fat, and dorsal subcutaneous fat. All data are presented as the mean and the standard deviation. Statistical analyses were performed using t-tests. Results were considered significant, if p<0.05.

Both the study using the normal diet (Table 1) and the study using the high-caloric diet (Table 2) show a significant decrease in adipose tissue mass in the treatment compared to the control group, while other organs such as, heart, lung, liver, spleen, and kidneys are not affected. The significant effect on the adipose tissue mass paired with the observation that other organs are not massively affected by this treatment demonstrates the feasibility of using an agonistic anti-LT-b-R antibody for the treatment of adiposity, as laid down in this application.

Figures Description

Figure 1 TNFSF14-Application rate/impact curve - murin

Figure 2 TNFSF14-Application rate/impact curve - human

Figure 3 Application rate/impact: Lta/b Heterotrimere on hLTBR

Figure 4 Proof of Receptor: mHVEM and mLTBR

Figure 5 Antibody-Application rate/impact: on mLTBR (curve)

Figure 6 Antibody-Application rate/impact: on hLTBR with pAK

(also HVEM)

Figure 7 Animal data with mTNFSFI 4

Figure 8 TNFSF14 and Glucose uptake

Figure 9 Zytotox TNFSF14 on 3T3-L1

Table 1 : Table LTBR-1 : Tissue weights (gram)

Table 2: Table LTBR-2: Tissue weights (gram)

Table LTBR-1:

Tissue weights (gram)

Table LTBR-1: Tissue weights. Data shown are mean ± standard deviation (n = 10). Results are considered significant if p< 0.05.

Young male C57BI/6 mice, fed a normal mouse chow diet, were injected with an lgG2a directed against LTB-R (50 μg/mouse i.p. three times weekly). Control mice were similarly injected with control isotype lgG2a. Study period was six weeks.

At autopsy, the weight of the epididymal fat pads and of the subcutaneous dorsal fat was significantly smaller in the mice treated with specific antibody. Treatment with an antibody against the LTB-R thus had significant effects on adipose tissue weights.

Table LTBR-2:

Tissue weights (gram)

Table LTBR-2: Tissue weights. Data shown are mean ± standard deviation (n = 10). Results are considered significant if p< 0.05.

Young male C57BI/6 mice, fed a high fat-diet for twelve weeks prior to treatment, were injected with an lgG2a directed against LTB-R (Group A; 50 μg/mouse i.p. three times weekly). Control mice (group B) were similarly injected with control isotype lgG2a. Study period was six weeks.

At autopsy, (abdominal and subcutaneous) adipose tissue weighed significantly less in the treated group, while the weight of other tissues did not differ. Thus, in male mice fed a high-fat diet for twelve weeks, treatment with an antibody against LTB-R resulted in significant weight loss of adipose tissue in the treated group.

LTBR-studies: Material & Methods for both experiments

Mice

Male C57BI/6J mice were housed during the experiment in clean-conventional animal rooms (relative humidity 50-60%, temperature ~21 ^βC, light cycle 6 am to 6 pm).

Where indicated high-fat diet contained 24% (w/w) lard (Hope Farms, Woerden, the Netherlands) or normal chow diet (normal rodent chow; Ssniff, Bioservices, Schaijk, the Netherlands) was given. Food and acidified water were given ad libitum. Mice were housed groupwise (5 per cage) in macrolon cages with bedding of wood chips.

Treatment

Rat-anti-mouse LTB-R lgG2a (Alexis, antibody 4H8 WH2, code ALX-804-145, lot L15482) and control rat lgG2a (R&D Systems) were used. Both preparations were diluted to 0.5 mg/mL with saline, and stored at 4⁰C. Mice were injected, three times weekly (Monday, Wednesday and Friday), in the morning, with 0.1 μl_ (50 μg) of a preparation intraperitoneal^.

Autopsy

After a 4-hr fast, mice were weighed, and then anaesthetized with FFM. The following tissues were weighed: heart, lungs, liver, kidneys, spleen, epididymal fat pads, perirenal fat and intestinal fat, and dorsal subcutaneous fat.

Data presentation

Data are presented in the report as mean ± s.d. Statistical analysis was performed using t-test Results were considered significant if p< 0.05.

Claims

Xantos Biomedicine AG 46EP34998FZ015pauTRANSLATED CLAIMS 1 to 23

1. Use of a lymphotoxin β receptor activating agent, preferably, a lymphotoxin β-receptor antibody (anti LT-β-R-AB), for the preparation of a pharmaceutical composition for the prevention and/or treatment of obesity and/or obesity associated diabetes (type II) and/or metabolic syndrome and/or other obesity related disorders.

2. Use of a lymphotoxin β-receptor activating agent according to claim 1 , wherein the agent imparts an activation of the lympotoxin β-receptor signal pathway.

3. Use of a lymphotoxin β-receptor activating agent according to any of claims 1 or 2, wherein the activation takes place alone or in combination with other lymphotoxin β-receptor activating agents.

4. Use of a lymphotoxin β activating agent according to any of claims 1 to 3, wherein the agent inhibits the differentiation of pre-adipocytes to mature adipocytes via a receptor mediated signal transduction and/or the storage of lipid molecules in differentiating and/or already mature adipocytes and/or promotes lipolysis.

5. Use of a lymphotoxin β-receptor activating agent according to any of claims 1 to 4, wherein the agent comprises an agonistic anti-LT-β-R antibody.

6. Use of a lymphotoxin β-receptor activating agent according to any of claims 1 to 5, wherein the agent is directed to a least one activating epitope of the lymphotoxin β-receptor.

7. Use of a lymphotoxin β-receptor activating agent according to any of claims 1 to 6, wherein the agent comprises polyclonal and/or monoclonal anti-LT-β-R antibodies.

8. Use of a lymphotoxin β-receptor activating agent according to any of claims 1 to 7, wherein the agent comprises functional variants and/or derivatives of anti-LT-β-R antibodies, in particular molecules with similar binding properties such as chimeric, recombinant, humanized or otherwise modified antibodies, antibody fragments, single domain antibodies and other specific binding proteins.

9. Use of a lymphotoxin β-receptor activating agent according to any of claims 1 to 8, wherein the agent comprises an agonist, in particular a protein with similar binding properties as antikalin, affilin or a non-protein such as aptamer.

10. A pharmaceutical composition comprising a β-receptor activating agent as defined in one of claims 1 to 9.

11. A substance, characterized in that it comprises at least one of the activating epitopes of LT-β-R.

12. Use of a substance according to claim 11 for the preparation of an activating LT- β-R antibody.

13. A pharmaceutical composition comprising a substance according to claim 11.

14. Use of a substance according to claim 11 for the preparation of a vaccine.

15. Use of a substance comprising an activating epitope of LT-β-R for the preparation of a vaccine for the prevention and/or treatment of obesity and/or obesity associated diabetes (type II) and/or metabolic syndrome and/or other obesity associated disorders.

16. A method for the selection of a LT-β-R activating agent comprising the step of contacting of the agent(s) which have to be tested with at least one substance comprising an activating epitope of LT-β-R and detecting of an interaction of the agent with the epitope.

17. A method according to claim 16, characterized in that the substance is expressed from a cell and the phenotype of the cell is determined in the presence or absence of said agent.

18. A method for the selection of an LT-β-R activating agent, wherein the activating of LT-β-R is effected by the binding of the agent to at least one activating epitope of LT-β-R and the method comprises the following steps:

a) sewing of undifferentiated pre-adipocytes and cultivating until post confluence,

b) incubating the cells with a specific adipocyte differentiating substance mixture and simultaneous incubating of the cells activated for differentiation with agents which are to be tested,

c) measuring of the level of differentiation of the cells, wherein the measuring comprises the detection of the level of the intracellular lipid,

d) identification of the fat cell differentiation modulating agent using the deviation of the test signal strength versus negative controls,

e) testing of the positively identified agent of step d) for specific binding to LT- β-R.

19. A method according to any of claims 16 to 18, characterized in that the cell is a mammalian cell, preferably of human origin.

20. A method according to any of claims 16 to 19, characterized in that the cell is a primary pre-adipocyte or a primary adipocyte or stems from primary pre- adipocytes or primary adipocytes.

21. A method according to any of claims 16 to 20, characterized in that the change of the biological activity of pre-adipocytes or mature adipocytes, preferably 3T3-L1- cell or human pre-adipocytes or adipocytes, is determined.

22. A method according to any of claims 16 to 21 , characterized in that the change of the biological activity consists of a inhibition or decrease of the fat storage in cells, preferably pre-adipocytes, differentiating adipocytes or mature adipocytes.

3. A method according to any of claims 16 to 22, characterized in that the fat storage is measured via determination of intracellular stored lipids.