DE10054279A1 - Use of ligands from death receptors or RIP to trigger caspase-independent cell death and compounds to inhibit caspase-independent cell death - Google Patents

Abstract

The invention relates to the use of physiologically occurring protein, in particular of death receptor ligands or RIP, to initiate the non-caspase-dependent cell death in immune system cells. The invention further relates to compounds, in particular also antibodies, which inhibit the initiation of the non-caspase-dependent cell death, with particular activity in inhibiting the signal transmission path by means of the protein RIP. The invention also relates to a method for identification of the above compounds.

Description

The present invention relates to the use of physiologically occurring proteins, especially of Ligands of death receptors (receptors with intracellu death domains) or RIP, to trigger the caspase independent cell death in immune system cells. Wei the present invention further relates to compounds in particular also antibodies that trigger the cas inhibit pase-independent cell death, especially inhi based on the signal transduction pathway via the protein RIP work. Finally, the present invention relates to Process for identifying the aforementioned Ver bonds.

It is well known that the death of cells on two can be done in different ways. For one, one can Cell undergo the process of "apoptosis", the Apoptosis is characterized by a shrinking of the be affected cell, through condensation of the nuclei and through internucleosomal degradation of DNA. (J. Kerr, A.H. Wyllie, A.R. Currie, Brit J of Canc 68, 239-257 (1972). To start which can result in the death of a cell by the phenomenon  the "necrosis" can be brought about. Fiers et al. (W. Fiers, R. Beyaert, W. Declercq, P. Vandenabeele, Oncogene 18, 7719- 30 (1999)) describe that the necrosis of an An swelling of the cell and organelles is accompanied, followed by cell disintegration with release of cytoplasmic components. In the publication by Fiers et al. it is also found that the cells krose Caspase-independent. Therefore, a caspa se-independent cell death in the literature mostly with the Equated phenomenon of necrosis.

Scaffidi et al. (C. Scaffidi, S. Kirchhoff, P. H. Krammer, M.E. Peter, Curr. Opin. Immunol. 11, 277-85 (1999)) shows that activation-induced cell death (activation- induced cell death (AICD) of T lymphocytes by the Zu interaction between FasL and TNF is triggered. In this Relation has been assumed to be the T cells through the expiration of the well-known caspase-dependent apopto program. Here the initiali runs apoptosis by stimulating the fascia Receptor after binding the Fas ligand (FasL). The in tracellular part of the death receptor Fas associates thereby with the adapter protein FADD, whereby the Binding of the Pro-Caspase-8 becomes possible and thus the Cell death-inducing signal complex (death-inducing si gnaling complex, DISC). In this complex the Pro-Caspase-8 is activated and in its active (mature) form released, causing apoptosis gently.

From the publications by Klas et al. (C. Klas, K. M. Debatin, R.R. Jonker, P.H. Krammer, Int Immunol. 5, 625- 30 (1993)) and Irmler et al. (Nature 388, 190-195 (1997)) is known in this context that significantly higher FasL concentrations are required to peri dormant killing phere blood lymphocytes (PBL) than T cells that for three days with phytohaemagglutinin (PHA) and IL-2 were activated.  

Vercammen et al. (D. Vercammen et al., J. Exp. Med. 188, 919-30 (1998)) could show that the Fas receptor is the Cell death also in a caspase-independent way, So in a supposedly necrotic way, then trigger can, if the Fas receptor in a highly sensitive fi brosarcoma cell line overexpressed and the caspase Activity is inhibited. It was also proven that cell death with necrotic morphology in caspase 8-deficient Jurkat cells can occur when an ar FADD is aggregated significantly (A. Kawahara, Y. Ohsawa, H. Matsumura, Y. Uchiyama, S. Nagata, J. Cell Biol. 143, 1353-60 (1998)).

From the prior art, however, apart from the Observation of morphological differences between the apoptotic and necrotic cell death, no system matic, the molecular mechanism of these two Va Boundaries related to cell death or over attitudes, especially with regard to the necroti cal signal cascade, known.

The object of the present invention is to develop strategies based on results from basic experiments on the molecular mechanisms triggering the necroti to develop (caspase-independent) cell death, d. H. especially native or artificial connections to make available that allow to enter the regulations tion of necrotic cell death and, if necessary, the necrotic cell death in specific medical indi to modulate cations.

According to the invention, it is now shown, surprisingly, that ligands from death receptors, for example TRAIL, FasL or TNF, i.e. the ligands of TRAIL-R, FasR or TNF-R, to trigger the caspase-independent, i.e. the non-apoptotic, cell death from cells, in particular Immune system cells, hepatocytes, heart muscle cells len or neurons and all other cells that make up the necrosis  by binding death receptor ligands gene, can be used (claim 1).

According to the phenomena of apoptosis and Necrosis summarized under the umbrella term "cell death". Apoptosis with the typical morpho described above Logical changes are dependent on Caspase, since the ex tracellular apoptotic signal via a cascade of Caspasen is passed on intracellularly. Apoptosis is therefore also called caspase-dependent cell death net. In contrast, the necrotic Va Riante of cell death independent of caspase. It will therefore defi in the present case also as caspase-independent cell death ned.

Thus, according to the invention, ligands of death become the first receptors also as an extracellular trigger of the necroti cell death, i.e. cell death with the typical mor phological manifestations of necrosis, especially in native, d. H. unmodified cells. in the Context of the present invention are under ligands of death receptors, especially among the ligands of FasR, TRAIL-R and TNF-R, both the membrane-bound nati ve ligand with transmembrane component as well as a soluble death receptor ligand (e.g. sFasL, sTRAIL or sTNF) understood without the transmembrane domain. Most notably preferred is the use of the ligands of TRAIL-R2 and TNF-R1.

In particular, the use of functional derivatives and / or alleles from FasL, TRAIL or TNF or other death receptor ligands for elicitation solution of caspase-independent cell death. Under Functional derivatives are, for example, N or C terminals shortened or extended, possibly also intrasequential modified by deletion, substitution or insertion Understanding FasL sequences that despite their sequence changes compared to the native form, its function as an exception Preserve caspase-independent cell death. typically,  the amino acid substitutions are conserved be of a native nature, d. H. for example an aromatic by a other aromatic amino acid, one polar by one their polar or a hydrophobic by another hydro phobic amino acid to be replaced. Preferably for purpose according to the invention such derivatives of recipe for death gate ligands are used which contain the necrotic Si trigger signal by binding to the respective receptors, which is at least that for binding to the receptors and the signal triggering have required domains.

Typically, the ligands are death receptors, especially Fast, TNF or TRAIL (or their functional le derivatives), to trigger a caspase-independent Cell death from lymphocytes, preferably peripheral blood lymphocytes (claim 2), especially in peripheral Blood lymphocytes from mammals and finally very special preferred for triggering a caspase-independent Cell death from activated human peripheral blood Lymphocytes, again preferred by activated human peripheral blood T-lymphocytes (claim 3) used.

In addition to using ligands from death receptors itself can also expression vectors that the nucleo tide sequence of the death receptor ligand or a function nelle derivative or allele of this ligand, for use in triggering the Caspase-independent Cell death, especially in cells of the immune system, ge long. The present invention therefore relates to also the use of such expressions according to the invention ons vectors as well as the use of cells (i.e. the Use to trigger the caspase-independent cell tods in particular cells of the immune system) that with death receptor ligands according to the invention or functional len derivatives of Ex containing death receptor ligands expression vectors are transfected. For the trans host cells used, it will generally mean to be mammalian cells, especially human cells Cells. In the case of medically indicated use  it will usually be the patient's autologous cells act ex vivo with an expression vector such as mentioned above, transfected and then the patient (re) transplanted. Through expression and secretion of the ligand from the (re) transplanted host cells the death receptor ligand can be its invention Function as a trigger of a necrotic, i.e. caspase unfold independent cell death in vivo.

In a preferred embodiment of the present Er the use of death receptor ligands, especially FasL, TRAIL or TNF (or derivatives here von), (for the manufacture of a medicament) for the treatment treatment of autoimmune diseases, in particular for treatment development of multiple sclerosis, TEN, diabetes, lupus erythe matosus or rheumatoid arthritis, then claimed, if the corresponding death receptor ligand with its Activity according to the invention as a trigger of the necrotic Cell death used in auto-aggressive immune cells comes (claims 4 and 5).

Among the members of the TNF ligand family that Lead cell death is the use of the ligand TNFα be particularly preferred, but also the use of TWEAK, Lymphotoxin-α, CD40, CD30 or CD27.

Furthermore, compounds according to the invention (Inhibitors) of the physiological function of a death re zeptor ligands, possibly also a combination of more than an inhibitor of the aforementioned type, for inhibiting the Causing caspase-independent cell death from cells of the immune system disclosed (claim 6). Such ver bindings (inhibitors) or compositions of such In hibitoren can be used as medicines. egg ne compound of the invention with inhibitory poten tial can bind to the ligand itself or to the or one of the extracellular domains of a death recipe dock the tors. In particular, the invention  Compound a fragment of a native ligand of a To be the receptor, the antagonistic the death receptor blocked, but does not trigger the physiological signal. Such an antagonist typically becomes a fragment the ligand's receptor binding domain.

The use of a / s according to the invention is preferred Compound (inhibitors) of physiological, necrosis ini Acting function of ligands from death receptors for the case that the inhibitor is an antibody against the ligand, especially the receptor binding domain of Ligand is directed (claim 8). An inventive Antibody typically becomes a monoclonal anti be body, possibly humanized, and can preferably also are present as a Fab fragment (claim 9).

An antibody according to the invention with the aforementioned in hibitorial properties can also be a sequence or sequence pattern or spatial structure on death Recognize the receptor itself and in this way bind of the physiological ligand completely or at least tw. prevent (competitive). Such an inhi bitter antibody can be provided which preferably to the binding domain of the recipe for death tors docks, but nevertheless the necrotic signal cannot induce.

However, organic molecules that block, for example, the interaction between the death receptor ligand, for example FasL, and its physiological death receptor, for example FasR, with the ability to intracellularly transmit the extracellular ne Crotic signal triggered by FasL also come as Inhibitors of the ligands or the function of the ligands according to the invention, as disclosed in the invention, into consideration (claim 10). Such organic chemical inhibitors will typically have a molecular weight <5000, in particular <2000, especially <1500 and be physiologically well tolerated. Possibly. they will be part of a composition with at least one further active ingredient and preferably auxiliaries and / or additives. The organic inhibitors according to the invention will particularly preferably have a binding constant of at least 10 ⁷ mol ^-1 . The chemical structure of the compound (of the organic molecule) according to the invention can, for example, be assigned either the property for binding to the ligand or to the death receptor.

Compounds according to the invention are preferred to one or more of the following ligands or the corresponding bind to receptors: TRAIL (TRAIL-R), FasL (Fas-R) and / or TNF (TNF-R) (claim 11). One is used Compound according to the invention for inhibi tion of a caspase-independent cell death from cells of the Immune system. A compound according to the invention (inhibitor interaction of ligand and necrosis Death receptor) can be used alone or in combination with wide active ingredients or pharmaceutical auxiliaries and additives are used as drugs. A compound according to the invention is preferred (Inhibitor) of the function of a death re according to the invention receptor ligands (i.e. inhibition of the triggering of the necro table cell death), e.g. FasL, (to produce a Drug) in the treatment of heart attack, Ge stroke or ischemic disease states or neurological diseases, for example Alzheimer's disease or Parkinson's disease, find use (claim 13).

Another object of the present invention is the use of the protein RIP ("Receptor Interacting Protein ") or a functional derivative (see following definition of a functional derivative) as intra cellular signaling molecule of a caspase-independent Si gnalkaskade to trigger the necrotic (Caspase independent) cell death (claim 14). RIP belongs to family of serine / threonine kinases. It has three domains on, namely an N-terminal kinase domain, a middle re domain and a C-terminal death domain (DD). While  according to the prior art it is known that the middle Domain and the C-terminal domain for the NF-kB Activation is important, is shown according to the invention that cells with RIP without functional N-terminal kinase Domain not subject to necrosis. I.e. the kinase Function of RIP is an indispensable part of the Phä notyps "necrotic cell death" and is used for transmission of the extracellularly induced Si gnals. Accordingly, the use of RIP or of a functional derivative of RIP as intracellular acting kinase, which is characterized by that downstream elements of the signal cascade to trigger solution of necrotic cell death, preferably via Phos phorylation of the same, be activated (claim 15).

In particular, therefore, the use according to the invention of functional derivatives and / or alleles of RIP for Triggering of caspase-independent cell death is disclosed. Functional derivatives include, for example, N- or C- Terminal shortened or lengthened, possibly also intrase quentiell by deletion, substitution or insertion mo understood RIP sequences that, despite their Se change of sequence compared to the native form its function as mediator of caspase-independent cell death four. Typically, the amino acid Substitutions are conservative in nature, i.e. H. e.g. one aromatic by another aromatic amino acid, ei ne polar by another polar or a hydrophobic be replaced by another hydrophobic amino acid. Possibly. can also the N-terminal kinase domain alone find use according to the invention.

Furthermore, the use of negati ven mutants (especially so-called double negative Mutants) of RIP for inhibition of caspase-independent Cell deaths revealed. With these RIP mutants, the ne Crotic cell death can be blocked as the signal continues line is interrupted. With these RIP mutants it is, for example, sequences which the N-  terminal kinase domain not or only incompletely (e.g. as a fragment). Bind such mutants through their death domain to the intracellular To desdomain of death receptors (or in the case of TNF to the corresponding adapter protein TRADD), but direct the Si gnal no further. The RIP mutant can also go visibly the kinase activity, for example. By mutations in active center, be inactive and so block the necrosis ren. In addition, the use of death domains revealed to block necrosis. These death domains can be, for example, fragments of proteins that are attached to the ne Participate in krotische signal transduction, for example FADD, TRADD or RIP. The Wei Transport of the signal interrupted.

In addition to the use of RIP according to the invention also expression vectors that contain the nucleotide sequence of RIP or a functional derivative or allele of RIP (or an infunctional derivative - as described above wrote) for use with the aim of Triggering (or blocking) the caspase-independent Cell death, especially in cells of the immune system, ge long. The present invention therefore relates to also the use of such expression vectors as well the use of cells according to the invention (i.e. ver to trigger (or block) the caspase independent cell death in particular cells of the immune system systems) with the inventive RIP or functional (or infunctional) derivatives of RIP containing Ex expression vectors are transfected.

According to claim 16 of the present invention, a compound is provided as a further object, which is characterized in that it inhibits the function of the RIP protein as an intracellular signaling molecule of a caspase-independent signaling cascade for triggering ne crotic cell death. This chemical compound is preferably an organic chemical compound with a molecular weight of <5000, in particular <2000, especially <1500 and is typically well tolerated physiologically (claim 17). Possibly. it will be part of a composition with at least one further active ingredient and preferably auxiliaries and / or additives and can be used as a medicament. The organic molecule will be particularly preferred if the binding constant for binding to RIP is at least 10 ⁷ mol ^-1 . The compound according to the invention will preferably be such that it can pass through the cell membrane.

The chemi according to the invention is very particularly preferred connection if it uses the kinase function of RIP hibiert (claim 18). Typically the connection with the physiological substrate of RIP kinase com petitively around the binding (phosphorylation) site in the N-terminal domain compete. Preferably, the che mix connection also non-competitive, e.g. B. by kova lent binding to an amino acid side chain in the substrate binding place, the natural substrate of the binding place displace. An invention is very particularly preferred appropriate chemical compound with specificity for the RIP Kinase, so it only inhibits this and no other ren kinases from the serine / threonine kinase family.

In a further preferred embodiment it is with chemical compound according to the invention, which RIP function according to the invention blocked to an oligopep tid that chemically modified (e.g. to lighten Passage through the cell membrane, especially through end permanent (especially N-terminal) sequence areas) can or not modified the phosphorylated Ab cut the primary sequence of a native substrate of RIP equivalent. For example. can an inhibitori according to the invention cal molecule, preferably a tetra to dodecamer, egg ne amino acid sequence corresponding to the native substrate contain or best from a native substrate sequence hen, which is preferably tetra to dodecamer typi also the sequence of the substrate  Has phosphorylation site. Possibly. can such a the oligopeptide may also be chemically modified, that the amide-like bond between the individual ami Resistant to proteolytic degradation te alternative chemical group (e.g. sulfur or Phosphorus bridges) is replaced.

In another preferred embodiment of the present The present invention is the compound of the invention anti-RIP antibody obtained in ex vivo or by genthera Human in vivo procedures introduced into host cells is and there is not secreted as "intrabody" son which can develop its effect intracellularly (e.g. Dopamine-secreting cells in Parkinson's patients subject to necrosis and now according to the invention such anti-RIP intrabodies are transfected). By such anti-RIP "intrabodies" are the cells before Protected necrosis.

According to the invention, it can be a chemical compound for the inhibition of caspase independent necrosis but also about an inhi act on the association of FasR and RIP. For example. can the chemical compound to one of the for the together storage (association) of FasR and RIP responsible Chen death domains (DD) bind and inhibit in this way act toric. The binding is preferably to the To domain of RIP, but it can also be an inhibi Dock the gate to the death domain of FasR, if necessary also on both death domains.

In the case of induction of necrosis via the death recipe ren Fas and TRAIL-R, especially TRAIL-R2, is also inhibition of the adapter protein FADD possible, the likewise for the forwarding of the necrotic Si gnals is required, but not as an adapter protein between the intracellular sections of each Receptors and RIP. Thus, a Ver binding as an inhibitor of necrosis also the related  Interrupt signal transduction by binding between the death domain of the death receptors, so about TRAIL-R or Fas, and the adapter protein FADD prevented is (ie binding a compound of the invention the receptor and / or FADD). The same applies to connections gene with inhibitory potential by binding to the Death effector domain from FADD and / or the death effector domain of the more downstream protein, the interacts with FADD.

A chemical connection according to the invention with the radio tion of blocking the necrotic function of RIP, can as a medicine. Find use. One is in particular chemical compound according to the invention (for the production a drug) for the treatment of diseases, for the at least tw. causal pathological hypernecrosis or symptomatic is suitable (claim 19). In order to can be a RIP inhibitor, especially an inhibitor of RIP kinase function or an inhibitor of association by Fas and RIP, especially for necrosis inhibition in the treatment of the following diseases or Manufacture of a medicament for the treatment of fol diseases are used: autoimmune crane kungen, heart attack, ischemic infarction, Alzhei disease mer or Parkinson's disease (claim 20). invention Compounds as RIP inhibitors are also used treatment of infectious diseases, especially viral infections fittings for use. In this way, the regular Cellular necrosis inhi observed in viral infections be the only release from the viruses Host cells and distribution in the host organism allowed.

Another object of the present invention are Methods ("screening methods") for the identification of Compounds with inhibitory properties in Hin look at the triggering or forwarding of signals, which eventually lead to cell necrosis, especially of Compounds that interact with a death receptor Ligands with a death receptor or the interaction of  RIP with a death receptor, an adapter potein, or egg blocked a native substrate of the RIP kinase. Possibly. can also the interaction of FADD with a death receptor in be hibited, especially in the event of the triggering of the Necrosis from FasL or TRAIL. According to the invention, it can is concerned with the connections to be identified Act substances that interact with the Death receptor ligand and the death receptor or the die RIP function as intracellular necrotic protein Block signal transduction cascade (e.g. the kinase function of RIP or the binding of RIP to the adapter molecule FADD).

Methods according to the invention provide that (a) cells, especially immune cells or hepatocytes, especially T-lymphocytes, are modified so that their apoptotic signal transduction pathway is blocked, (b) these cells are made available according to (a) modified cells in a cell culture , (c) test substances are added to the cell culture, (d) death receptor ligands, in particular FasL, TRAIL or TNF, are added to the approach according to (c) and (e) the viability of the cells in the cell culture is determined (claim 21) , In an alternative procedure, however, the sequence of process steps (c) and (d) can be interchanged, so that the death receptor ligand is first added to the cell culture and only then is the test substance added. According to process step (d), concentrations of death receptor ligand are typically added, which trigger the necrosis in the cells. In the final process step (e), the effect of the test substance added is determined using the number of surviving (not subject to necrosis) cells. For this purpose, several parallel experiments with increasing concentrations of the test substance are preferably carried out in accordance with the method according to the invention in order to be able to determine the ID ₅₀ value in the event of a necrosis-inhibiting effect of the test substance.

A "screening" method according to the invention can also be carried out with With the help of so-called "proteomics" techniques become. First, the expressed protein for example on apoptosis inhibition with zVAD deleted cells (e.g. Jurkat cells) after adding (1) Death receptor ligand or (2) death receptor ligand and test substance (e.g. via 2D gel electrophoresis) Right. About procedures that make up the differences in the respective expression patterns, the effect samness of the test substance with regard to inhibition the necrosis is determined. Beforehand to determine a Standard typical differences in the expression pattern non-necrotic compared to necrotic cells collected experimentally.

The pretreatment of the cells according to process step (a) allows the effectiveness of the test substances (necrosis Inhibition) on the cells without any superimposed apop investigate dead events. The blockade of the apoptotic signal transduction can be formation of caspase-deficient cells, especially immune cells len, especially of Caspase-8 deficient immune cells, be ensured (claim 22). This is the one Caspase-dependent apoptotic signal transduction switches, so that by death receptor ligands, esp special FasL, TIRAIL or TNF, induced signals alone affecting necrosis. This also includes the use of Caspase-deficient (especially Caspase-8 and Caspase- 3-deficient) immune cells, especially peripheral T- Lymphocytes, to identify necrosis inhibitory compounds disclosed.

In an alternative procedure, the blockade of the apoptotic signal transduction path according to (a) by pretreating the cells, especially immune cells or hepatocytes, with one or more caspa se inhibitors, for example zVAD, can be achieved (claim 23).  

Finally, a method for Identification of necrosis inhibiting compounds also with the help of such cells, especially immune cells len are carried out in terms of function of the adapter protein FADD deficient (claim 24). Because FADD not only on the routing of the signal to the FasL or TRAIL induced necrotic (caspase independent) cell death is involved, but also in the FasL or TRAIL induced apoptosis, FADD- deficient (immune) cells only in the case of the invention Procedures are used when the signal doesn't go over the death receptors Fas and TRAIL-R (especially TRAIL- R2) is forwarded. According to this Procedures for ligands from other death receptors added according to process step (d), for example TNF. In order to the use of FADD will also be deficient Inhibit (immune) cells to identify necrosis the connections disclosed.

The present invention is illustrated by the following Figures explained in more detail:

In Fig. 1 it is shown that the FasL and activation-induced cell death in primary human and mouse T cells is insensitive to the addition of the caspase inhibitor zVAD-fmk.

Fig. 1a illustrates experiments represent, in which peripheral blood T lymphocytes (PBL) with PHA and IL-2 for each represents asked exposure time ( "resting", ie tion without activa, 24 h and 72 h) were treated and then for the duration of four hours with cross-linked ("cross-linked") soluble Fas ligand (FasL) in the presence (⚫) or absence (○) of zVAD-fmk (concentration 50 µM) were incubated. The FasL concentrations in ng / ml are plotted on the x-axis and the percentage of surviving cells on the y-axis. Significantly higher concentrations of FasL have to be used to induce cell death when the PBLs are at rest (left application) than with PBLs after three days of activation with PHA and IL-2 (right application). The addition of the caspase-x inhibitor zVAD-fmk (inhibits all caspases) only leads to a weak inhibitory effect in the case of dormant PBLs. In addition, an inhibitory effect cannot be observed with activated PBLs (middle and right application).

Referring to FIG. 1b splenocytes (T-cells of the spleen) for a period of three days with Concanavalin A IL-2 and ak tivated and then subjected to a twelve-hour incubation with FasL for triggering of cell death. The specific cell death for the CD4 ⁺ subpopulation was calculated. Here, too, it can be seen that, despite the addition of zVAD-fmk (∎), cell death can be observed, which means that caspase-independent cell death.

Fig. 1c shows the result from experiments with de nen PBL five days were activated and were then cultured for one to sätzlichen day only in IL-2, and finally re-stimulated with PHA, thereby induced cell death tion by activa (AICD) in To induce the presence or absence of zVAD-fmk. That in this case too, as can already be seen from FIG. 4a for FasL stimulation, the signal transduction, which is responsible for cell death, at least in part. Caspase-independent it follows from the results of the experiments after or without the addition of Caspase-x inhibitor zVAD-fmk. Here, too, it is shown that the aforementioned inhibition of the caspases has no significant effects on the number of specifically killed cells.

According to FIG. 1d, BALB / c mice PBS (for control) or SEB (150 μg) were injected into the foot paw. The mice were sacrificed after three days. Then the splenocytes were cultured for 24 hours before the addition of FasL (1 µg / ml). The specific cell death of Vβ8 ⁺ T cells (in activated form) and Vβ8 ^- T cells (non-activated cells) was compared. The results given correspond to the average of three measurements and are representative of at least three independent test series.

It is clear from FIG. 2 that FADD is required for FasL-mediated, caspase-independent cell death in T cells. T cell clones and transformed T cell lines were treated for 16 hours with the specified concentrations of FasL and the survival rate was investigated with a cell proliferation assay.

In Fig. 2a it is shown that the killing of T cell lines or T cell clones by Fas is partly caspase-independent. A stimulation-dependent human CD4 ⁺ T helper cell clone (left application), a human Jurkat T cell line (middle application) and a BJAB-B lymphoma cell line (right application) were used with the concentrations of FasL indicated on the x-axis and zVAD-fmk (0 (), 5 (⬩), 10 (⚫), 25 (▲), 50 (∎) and 100 () µM). The percentage of surviving cells (OD at 490 nm) is plotted on the y-axis. In all T cell clones or transformed T cell lines (left and middle plots), the protective effect that inhibits cell death, which is achieved by adding zVAD-fmk against the factor FasL that triggers cell death, can be seen. In these cases, Fas triggers the apoptotic signal transduction pathway to a significant degree - in contrast to the situation with primary T cells.

Nevertheless, the T cell lines die even when Solutions with highly saturated concentrations of zVAD-fmk (<25 µM). To rule out the observation of the Cell death in the T cell lines due to an inability of the Cas pase inhibitor zVAD-fmk has to be returned completely block cellular caspase activity, was investigated whether a residual activity of caspase-3 in  dying cells can be found. Since no pro teolytic activity of caspase-3 was detected, the T cells have the property of caspase to be able to find cell death independently through the FasL effect NEN.

In Fig. 2b (explanation of the symbols used to indicate the concentration of zVAD in Fig. 2a), the results of experiments with tumor cell lines are shown. It can be seen that - in contrast to T cell lines - the tumor cell lines show no cell death phenomena even at high concentrations of zVAD, ie the cell death of the tumor cell lines is already with small additions of zVAD-fmk to suspensions of tumor cell lines (e.g. the B-cell lymphoma cell line BJAB (left application) or the Adeno carcinoma cell line HeLa (right application)) largely blocked. Caspase-independent cell death events therefore do not take place in the tumor cell lines shown in FIG. 2b.

Further experiments shown in FIG. 2c also prove that a loss of caspase-8 activity is not sufficient to inhibit Fas-induced death signals. The human CEM-T cell line or a stable CrmA transfectant from this (left-hand plot) and Jurkat T-cells (right-hand plot), which are either caspase-8 deficient or not, were analyzed with FasL with or without zVAD-fmk (50 µM) treated. The plot on the x and y axes corresponds to the rest of the explanations for Fig. 2a. The left-hand plot of FIG. 2c shows the results from experiments with the wild type without the addition of caspase inhibitors (& ≎≄⊈≆↪⊲∵), with wild type with the addition of the general caspase inhibitor zVAD-fmk (∎) Addition of the viral protein and caspase-8 inhibitor CrmA (○) and when combined addition of CrmA and zVAD-fmk (⚫). Cas pase-independent cell death events are observed both when incubated with zVAD-fmk and with CrmA.

From the right-hand plot of FIG. 2c, it is clear that a Caspase-8 deficient Jurkat T cell line is also subject to cell death, namely at FasL concentrations which are of the same order of magnitude as for cell death in the presence of the inhibitors zVAD-fmk or CrmA are required.

Fig. 2d (axis plot see Fig. 2a) shows that the signal of the FasL-induced, caspase-independent cell death is passed on through the FADD death effector domain. Jurkat T cells that are deficient in FADD or not, as well as CEM T cells or a clone stably expressing the FADD death effector domain were treated with FasL with or without zVAD-fmk (50 μM).

The left-hand plot of FIG. 2d illustrates that Jurkat wild-type cells () from about 5 ng / ml FasL are subject to cell death. By contrast, no cell death can be observed in FADD-deficient Jurkat cells (∎), not even at high FasL concentrations. FADD thus proves to be an important signal transduction protein for both caspase-dependent and caspase-independent cell death.

The right-hand plot of FIG. 2d shows the results of experiments with wild-type (WT) -CEM-T cells and CEM-T cells, which stably overexpress a dominant, non-functional variant of FADD (FADD-DN). FADD-DN shows has a death domain, but does not have a death defect domain (DED). Compared to the situation in the wild-type (), the stable FADD-DN over-expressing CEM cells (o) prove to be much better protected against FasL as a cell death trigger. Since the survival rate of the FADD-DN cells is also significantly higher than that for the wild type with the addition of the caspase inhibitor zVAD-fmk (∎) (ie in this case the cells can no longer depend on the caspase, but can still do so on the caspase independent cell death), FADD's death effector domain (DED) is required for both caspase-dependent and caspase-independent signal transduction. As expected, the addition of the Cas pase inhibitor zVAD-fmk to FADD-DN cells (⚫) does not change the cell death behavior further depending on the FasL concentration.

In Fig. 3 is illustrated that the protein RIP binds to Fas and thereby the necrotic T cell death is triggered. The survivability assays were carried out as described in the corresponding exemplary embodiment.

Fig. 3a shows that RIP-deficient Jurkat T cells are resistant to FasL-induced cell death in the absence of Cas pase activity (with the addition of 50 uM zVAD-fmk, (⚫)). The measurement curve shows no drop (which would indicate the occurrence of cell death events), not even at high FasL concentrations. In the absence of zVAD-fmk, the wild-type Jurkat cells () and the RIP-deficient Jurkat cells (○) are equally sensitive to FasL apoptosis induction.

In Fig. 3b (axis plot see Fig. 2) it is shown that wild-type Jurkat cells are completely resistant to the triggering of caspase-independent cell death when they no longer have any rip protein. Results of experiments are shown in which Jurkat T cells were treated for 14 hours with geldanamycin, an HSP-90 inhibitor, at the stated concentrations and then with FasL for 16 hours in Present: from zVAD-fmk (50 µM) (to suppress any caspase-dependent cell death) and cycloheximide (2 µg / ml) were incubated. Geldanamycin inhibits Hsp90 and can therefore indirectly break down cellular RIP. Increasing concentrations of Gelda namycin (0 (Δ), 0.05 (○), 0.25 (◊) and 1 () µg / ml) reduce the cellular fraction of RIP, so that the wild-type Jurkat cells finally at a concentration of 1 µg / ml geldanamycin also proved to be insensitive to caspase-independent, FasL-induced cell death.

The respective expression levels of Fas, FADD, Caspase- 8 remained unaffected during RIP expression this pretreatment with geldanamycin depends on the concentration gig decreased (right illustration). With a concentration of 1 µg / ml geldanamycin is no longer a RIP protein weisbar.

According to FIG. 3c, the RIP-mediated caspase-independent cell death is independent of the protein synthesis. To assign this, Jurkat T cells were treated with the protein synthesis inhibitor cycloheximide (CHX, 2 µg / ml) and FasL for 16 hours and the cell viability was measured. It can be seen in Fig. 3c, that the addition of cycloheximide promoted cell death (∎) (compared to the mixture without cycloheximide (), the cell death is not therefore inducing property of RIP to its ability, the expression of NF-kB to induce can be returned.

From FIGS. 3d and 3e it can be seen that RIP-deficient Jurkat cells become capable by transfected wild-type RIP, but not by RIP kin dead or kinase-deficient RIP (RIP Δkin) Recover FasL-induced, caspase-independent cell death. The N-terminal kinase domain of RIP is therefore required for the induction of caspase-independent cell death. After electroporation of RIP-deficient cells with control vectors or plasmids, either wild-type RIP or mutant RIP was expressed. After 24 hours, the cells were analyzed with zVAD-fmk (50 μM) with increasing concentrations of FasL for 16 hours and finally with regard to the specific cell death (propidium iodide (PI) labeling) by flow cytometry.

The expression rates of RIP proteins in transfected cells are shown in FIG. 3e as an excerpt from a "Western blotting" plot (small diagram with details in kDa on the right). The data sets given correspond to a representative value of 7 mutually independent experiments. In the right illustration of FIG. 3e is the percentage of PI-positive cells for an experiment, were fied in the Jurkat cells with a GFP ( "green fluorescence protein") expression plasmid Cotrans. The analysis is based solely on selected GFP-positive cells and shows the respective proportion of cells in the total amount in the form of bar diagrams for -FasL (left) and + FasL (right).

Referring to Fig. 3f, the fast induced is time-dependent Ag glomeration the death signaling complex (DISC) in wild type (either with zVAD-fmk (50 pN) treated or not treated) and FADD deficient Jurkat cells Darge provides. The complexes (DISCs) were immunoprecipitated (IP) using flag-labeled FasL or non-agonist anti-Fas antibody (ZB4) and for the presence of Fas, FADD, Caspase-8 and RIP by immunoblotting techniques was investigated. The experiments shown correspond to a representative cross-section of at least three independent test series. Rapid, time-dependent mobilization of Fas, FADD, pro-caspase-8 and significant amounts of RIP can be observed. In contrast, no binding of RIP to Fas is detectable if the immunoprecipitation is carried out with a non-agonistic anti-Fas antibody (application on the far right in FIG. 3f). This proves that the binding of RIP to Fas can only take place after appropriate stimulation. Since the binding of RIP to Fas can also be observed in FADD deficient cells, RIP is bound directly to Fas without mediation of the adapter protein FADD.

FIG. 4 shows the FasE-induced, caspase-independent T cell death, in conjunction with necrotic morphological changes which are independent of a cytochrome c release.

In Fig. 4a are electron micrographs of Jurkat cells incubated with and without FasL (1 ug / ml) for a period of four hours is illustrated. The bar corresponds to one micrometer. Jurkat cells that had been treated with FasL show the typical apoptotic features, for example the temporary chroma tin condensation (middle illustration). In contrast, Jurkat cells after treatment with zVAD-fmk (to switch off caspase-dependent cell death, right-hand illustration) or Caspase-8-deficient Jurkat cells have neither the phenomenon of chromatin condensation nor oligonucleosomal DNA degradation.

From Fig. 4b it can be seen that necrotic cell death is characterized by damage to the plasma membrane and by surface exposure to phosphatidylserine (PS). The damage to the membrane (propidium iodide influx, PI label) and the PS translocation (Annexin V label) was determined at various times after the addition of FasL (1 µg / ml) in Jurkat cells (left illustration) and examined in PBL activated for five days (right illustration), namely in the presence ((⚫), (∎)) and in the absence ((o), ()) of zvAD-fmk. As expected, no membrane damage was observed in apoptotic Jurkat cells even after 6 h (o), while phosphatidylserine exposure on the cell surface could be observed relatively quickly ((), less than 100 min). In contrast, a continuous increase in PS translocation (∎) and membrane damage (⚫) can be observed during the observation period with the addition of caspase inhibitor zVAD-fmk. The PS translocation starts before the membrane is damaged. With activated PBL (right illustration), a considerable proportion of necrotic cells can be observed even in the absence of caspase inhibitors due to the measured membrane damage (o). In the presence of zVAD-fmk, PS translocation and membrane damage are faster and more pronounced with activated PBL than with the Jurkat cells used for comparison ((∎), (⚫)).

In Fig. 4c that RIP in PBL in all phases of activation by PHA / IL-2 is highly expressed is shown. The expression of Fas, FLIP (L), FLIP (S) and FADD, determined with the aid of immunoblotting techniques, served as a control.

From Fig. 4d it follows that a reduction in the mito chondrial membrane potential ψm in FasL-treated Jur kat cells can be observed. Typically, the mitochondrial membrane is permeabilized in apoptotic cells, whereupon the mitochondrial membrane potential decreases. The decrease in membrane potential ψm against time is plotted in min. In the absence of zVAD-fmk (), the onset of this course, which is typical of apoptosis, can already be observed after 10 min. In contrast, FasL-induced necrosis (the apoptosis is switched off by adding zVAD-fmk) only after 4 h to a depolarization of the mitochondrial membrane, i.e. at a point in time at which the necrotic cells are already undergoing considerable morphological changes (∎) ,

Fig. 4e shows, inter alia, the cytochrome c release from mitochondria which occurs in apoptotic, but not necrotic Jurkat T cells. For this purpose, the Jur kat cells were treated with FasL as described above, and the cytochrome c expression was measured after 0, 15, 30, 60 120 and 240 min. The cell extracts were separated into their cytoplasmic (S / N) and into their nuclear mitochondrial (P) components and examined for the presence of cytochrome-c with the help of immunoblotting techniques. It can be clearly seen in the left half of FIG. 4e that the amount of cytochrome c from the P fraction decreases over time, but increases correspondingly in that in the S / N fraction. As is typical for apoptotic cells, after the signal cascade is triggered by FasL, cytochrome flows into the cytoplasmic fraction. In contrast, in Jurkat cells, which cannot become apoptotic due to the addition of zVAD-fmk, but which become ne crotic due to FasL-Wikung, the amount of cytochrome c in the P fraction remains constant during the observation period, so that in Cytoplasm no cytochrome-c can be detected.

The progress of apoptotic cell death could be there by determining that the typical for apoptosis Caspase-8, Caspase-9, Caspase-3, Caspase-2, Caspase-7, PARP (poly-ADP-ribose polymerase) - and bid-cleavage in the cytoplasmic fraction was checked (left panel supply). All of the aforementioned proteins are subject to the for Apoptosis typical activation, i.e. the proteolytic Conversion from the inactive to the active form. On the other hand show those treated with zVAD-fmk and therefore the apopto se inaccessible, necrotic Jurkat cells neither an activation of the aforementioned caspases by their proteolytic cleavage still cleaves the protein Bid.

A typical further characteristic of apoptosis, namely the translocation of CAD / DFF40 into the nuclear fraction, cannot be determined in the case of cell necrosis caused by FasL (likewise shown in FIG. 4e). In addition, neither cytochrome release from the mitochondria nor cleavage of the caspase-3 substrate PARP can be observed, even over an observation period of 4 hours.

Fig. 5 presents plots of experiments showing the viability of various cells after adding TNF (TNFα) or TRAIL. The percentage of surviving cells is plotted as a function of the concentration of the ligand added, the following conditions being selected for the test batches (with DMSO (), with zVAD (50 µM, ∎), with CHX (2 µg / ml, o) or with CHX / zVAD (⚫).

Fig. 5a shows the corresponding representation of experiments with Jurkat cells (WT) with the addition of TNFα. The cytotoxic effect of TNF was significantly reduced by adding zVAD-fmk (∎). In contrast, pretreatment with cycloheximide (CHX) leads to cell death in Jurkat cells even at concentrations of only 50 ng / ml, which is independent of the caspase function (⚫). Pretreatment with CHX alone without the addition of zVAD only slightly increases the cytotoxic effect, so that as a result the phenomenon of necrotic, caspase-independent cell death - in this case by induction of TNFα on the receptor TNF-R1 - when protein synthesis is switched off CHX runs particularly efficiently. In the right-hand plot of FIG. 5a, the effect of the ligand TRAIL Todesrezeptor- to WT-Jurkat cells is retained. After pretreatment with CHX, a clear induction of necrosis can also be observed (via the TRAIL-R2 receptor).

In Fig. 5b is the percentage of viable Jurkat cells RIP-deficient are, after exposure to TRAIL (right) and applied TNFa (left). Necrotic cell death is not observed in this case when TNFα is added. (∎). This also applies to the effect of TRAIL. The experiments shown in FIG. 5b show that the function of RIP is indispensable for the intracellular transmission of the necrotic signal.

From Fig. 5c shows that TNF is able to induce necrosis in FADD deficient cells. The absence of FADD can even drive the cells into caspase-independent cell death per se. In contrast, TRAIL cannot trigger necrotic events in FADD-deficient Jurkat cells (not shown). This means that there are differences with regard to the presence of the FADD adapter molecule - depending on which death receptor ligand (FasL, TRAIL or TNF) triggers the signal cascade.

Accordingly, according to the findings according to the invention, FIG. 6 shows a schematic model for the signal cascade in necrosis, in contrast to apoptosis. FasL and and TRAIL use the adapter proteins FADD and RIP for signal transmission via their respective receptors Fas and TRAIL-R2. In the case of apoptosis (shaded in dark), FADD binds to the corresponding intracellular domain of the receptor via the death domain and to caspase-8 via the death effector domain. In the case of necrosis, FADD and RIP bind to the receptor. RIP phosphorylates another, previously unknown protein downstream, FADD binds to another protein of a previously unknown nature.

In contrast, is for necrotic signal transmission after induction via TNF (and e.g. TNF-R1) FADD not he required, but only the adapter protein TRADD, which the Contact from RIP to the intracellular part of the receptor taught.

The present invention is illustrated by the following Exemplary embodiments explained in more detail.

1st embodiment (a) Purification and analysis of human T cells

The T cells were purified from human PBL by negative selection using the miniMACs magnetic cell separation system. For this purpose, the cells were incubated on ice with anti-CD11b and anti-CD20 monoclonal antibodies in order to eliminate natural killer cells or B cells, followed by goat anti-mouse IgG, coupled to magnetic beads ("beads"), before they were loaded on a separation column. More than 95% of the cells isolated were CD3 ⁺ . The purified T cells were cultured for a further 24 hours in complete RPMI-1640 medium containing 5% human serum. Peripheral blood lymphocyte PBLs were activated with PHA (0.5 µg / ml) and IL-2 (20 U / ml), and sensitivity to FasL was increased after 20 minutes preincubation with zVAD-fmk (50 µM) or solvent (DMSO ) tested. The activated cells were distributed on 96-U "well" microtiter plates with serially diluted cross-linked sFasL (from Alexis). After 4 hours of incubation, the cells were labeled with Annexin V (AnnV, from Nexins Research) and propidium iodide (PI) according to the manufacturer's instructions. The specific cell death was calculated as a decrease in the double negative population, based on the untreated control.

(b) Activation-induced cell death

The PBL were kept in PHA (0.5 µg / ml) / IL-2 for 5 days (20 U / ml) activated, washed and for wide re 36 hours in medium, supplemented only with IL-2, cult fourth. The cells were then treated with PHA in the presence of zVAD-fmk (50 µM) or solvent for 16 Hours restimulated and death was as described above wrote, measured by AnnexinV / PI marking. The right Lative activation-induced cell death was derived from Experi elements that were performed in triplicate as follows calculates: (1- (cells treated with PHA / untreated Cells)) x 100.

(c) Analysis of mouse T cells

Splenocytes from 8 week old BALB / c mice were for three days in the presence of Concanvalin A (2 µg / ml) cultivated, for 20 minutes with zVAD-fmk or solvents preincubated and towards cross-linked human FasL added. After 8 hours of treatment, the cells resuspended in FACS buffer and with anti-CD4-CyChrome marked, then marked with AnnV and with the help of flow cytometry analyzed.  

BALB / c mice were 150 µg from SEB (from Alexis) or PBS (Control) injected into the rear paw; the mice were sacrificed after three days. The splenocytes were homogenized in a medium through a sterile steel sieve and then separated from the remaining particles by sedimentation riert. The cells were cultured for 24 hours before the surviving cells were treated with FasL were. The lymphocytes were washed in FACS buffer, labeled with biotinylated anti-VβB, anti-CD4-CyChrome and anti-CD8-PE for 15 minutes, then with AnnV and Streptavidin-AFC marked and with the help of Flow cytometry analyzed. The results were like calculated as follows: (% AnnV positive cells treated with FasL) - (% AnnV positive cells in control).

The results of the first exemplary embodiment are shown, for example, in FIG. 1 (a to d) and are described in the associated description of the figures.

2nd embodiment Cytotoxic tests

To carry out the cytotoxic test series, cells were preincubated for 20 minutes with zVAD-fmk or solvent and then to titrated human sFasL, sTRAIL and sTNF (sold by Alexis) in 96 "well" microtiter plates to a final volume of 100 µl added (in the case of HeLa cells, 0.8 µg / ml of cycloheximide was added in all test series). The activity of sFasL and cross-linked sTRAIL (called "TRAIL" in Fig. 5) was enhanced by the addition of a 1 µg / ml cross-linking anti-flag antibody (M2, Sigma). After 16 hours of incubation, the test system was mixed with 20 ul PMS / MTS reaction mixture (from Prome ga) and, as soon as a corresponding contrast had developed, the optical density for the individual plates was determined at a wavelength of 490 nm.

The results of these experiments are shown, for example, in FIGS. 2 and 5 and are described at the appropriate place.

3rd embodiment DISC analysis

Jurkat cells were exposed to 2 µg / ml FasL or Absence of cross-linking anti-FLAG-M2 antibody (2 µg / ml) in the time interval shown in the figures vallen treated. The cells were then quickly run through the addition of 5-fold volumes of ice-cold PBS cool, then with 0.2% NP-40, 20mM Tris-HCl (pH 7.4), 150mM NaCl, 2 mM sodium vanadate, 10% glycerol and the Protea inhibitor cocktail lysed. The cytosolic fraction were pre-cleaned with Sepharose 6B for 90 minutes and then with protein G-coupled Sepharose beads incubated for 3 hours. The bead was then washed four times with the lysis buffer. The pro tones were then separated on SDS-PAGE and opened Nitrocellulose filter "blotted". In the corresponding Control experiments became Fas with non-agonistic anti-Fas antibody ZB4 (MBL) immunoprecipitated. To De detection of the individual proteins (Fas, FADD, Caspase-8 and RIP were the following antibodies for the "Western Blotting "uses: anti-Fas (from Santa Cruz), anti-FADD (from Transduction Labs), anti-Caspase-8 (from MBL).

The results of these experiments are shown in Fig. 4f and the associated description.

4th embodiment Reconstitution of RIP-deficient cells

For the reconstitution of Rip-deficient Jurkat cells, the cells were diluted to a density of 250,000 cells / ml and cultured for one day. For the transfection, the cells were washed and then resuspended at a density of 12.5 × 10 ⁶ cells / ml. 800 µl of the cell suspension were mixed with a total amount of 30 µg of the plasmid DNA, which in addition to a carrier contains a plasmid which codes for the large T antigen (5 µg) and 5 µg of the respective RIP expression plasmids. After electroporation, the cells were cultured in medium with additives, supplemented by zVAD (50 μM), for a period of 24 hours. Half of the culture was used to control the expression levels (by Western blotting), the rest of the cells were added to FasL in the presence of zVAD (50 μM) and incubated for 14 hours. The cells were labeled with 5 µg / ml PI and analyzed immediately. In some experiments, a plasmid coding for GFP ("Green Fluorescence Protein") (1 ug) was transfected into the RIP-deficient Jurkat T cells - together with the plasmids mentioned above. The cells were separated by FACS and the analysis was carried out selectively on GFP-containing cells.

The results of these experiments are shown, for example, in FIGS. 3d and 3e and in the associated description.

5th embodiment Biochemical and morphological analysis of necrotic cell

The cells were zVAD for 15 minutes (50 µM) preincubated and after the specified time periods  treated with FasL. Half of the cells were in 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.2% NP-40, ent holding protease inhibitors, lysed and then centrifuged yaws. The lysates were designed for activation from Caspase-8 (MBL), Caspase-9, Caspase-3, Caspase-2, Bid (Transduction Laboratories) PARP and Caspase-7 (Biolabs) analyzed with the help of "Western blotting".

With regard to cytochrome c release, the cells were incubated for 10 minutes on ice in 20mM Hepes pH 7.5, 10mM KCl, 15mM MgCl ₂ , 1mM EDTA, 1mM EGTA containing protease inhibitors then lysed with 15 impacts in a syringe (gauge G22). The lysate was incubated on ice for 10 minutes and then centrifuged. The supernatants and pellets (precipitates) were resuspended in reducing Laemmli buffer and tested for the presence of cytochrome-c (Pharmingen).

The results of these experiments are shown, for example, in FIG. 4 and the associated description.

Claims (24)

1. Use of a ligand of a death receptor or a functional derivative of the same for triggering of a caspase-independent cell death of cells, ins special of cells of the immune system. 2. Use of a ligand of a death receptor or a functional derivative thereof according to claim 1 to trigger caspase-independent cell death of peripheral blood lymphocytes. 3. Use of a ligand of a death receptor or a functional derivative thereof according to claim 1 or 2 to trigger a caspase-independent Cell death from activated human peripheral blood T- Lymphocytes. 4. Use of a ligand of a death receptor or a functional derivative of the same after a of claims 1 to 3 for the manufacture of a medicament means for the treatment of autoimmune diseases. 5. Use of a ligand of a death receptor or a functional derivative of the same after a of claims 1 to 4, wherein the ligand FasL, TRAIL or is TNF. 6. Connection, characterized in that it belongs to the Ligands of a death receptor binds and thus the Triggering a caspase-independent cell death from Cells, especially cells of the immune system, inhibited. 7. Connection according to claim 6, characterized in that it is an antibody against the ligand of the death receptor.   8. A compound according to claim 6 or 7, characterized indicates that it is a monoclonal antibody. 9. Connection according to one of claims 6 to 8, characterized characterized as being a humanized antibody is. 10. A compound according to claim 6 or 7, characterized records that it is an organic chemical compound with a molecular weight of <1500. 11. Connection according to one of claims 6 to 10, there characterized in that they attach to the ligands TRAIL, TNF or FasL binds. 12. Use of a connection according to one of the claims 6 to 11 to inhibit the release of a caspase independent cell death from cells, especially from Immune system cells. 13. Use of a compound according to one of the claims 6 to 11 for the manufacture of a medicament for loading act of autoimmune diseases, heart attack, ge stroke or neurological diseases, e.g. Alzheimer's disease or Parkinson's disease. 14. Use of RIP or a functional derivative of RIP as an intracellular signaling molecule of an Si gnalkaskade to trigger the Caspase-independent Cell death. 15. Use of RIP or a functional derivative of RIP according to claim 14 as an intracellular kinase for phosphorylation of downstream elements of a Signal cascade to trigger the caspase-independent Cell death. 16. Connection, characterized in that it is the Function of the RIP protein as an intracellular signaling molecule  a caspase-independent signal cascade inhibited to trigger cell death. 17. A compound according to claim 16, characterized in that they have an organic chemical connection with egg nem molecular weight of <1500. 18. A compound according to claim 16 or 17, characterized records that it is the kinase function of RIP inhi biert. 19. Use of a connection according to one of the claims 16 to 18 for the manufacture of a medicament for Treatment of diseases for which at least tw. a pathological hypernecrosis causally or sympto is matical. 20. Use of a compound according to claim 16 to 18 for the manufacture of a medicament for treatment of autoimmune diseases, heart attack, ischemic Infarction, Alzheimer's disease, Parkinson's disease or vi infectious diseases. 21. A method for identifying compounds which act as inhibitors of caspase-independent cell death, in particular compounds according to one of claims 6 to 11 or 16 to 18, characterized in that

• a) cells, especially immune cells, in particular T lymphocytes, are modified so that their apoptotic signal transduction pathway is blocked,
• b) these cells are provided in a cell culture modified according to (a),
• c) test substances are added to the cell culture,
• d) death receptor ligands, in particular FasL, TRAIL or TNF, are added to the approach according to (c) and
• e) the viability of the cells in the cell culture is determined.

22. The method according to claim 21, characterized in that in process step (a) caspase-deficient (Immune) cells, especially caspase-8 deficient ones (Immune) cells are produced. 23. The method according to claim 21, characterized in that that the cells according to (a) one or more caspase Inhibitor (s) are added. 24. The method according to claim 21, characterized in that in process step (a) FADD-deficient (Immune) cells are produced and used in process step (d) TNF is added.