NO321514B1 - DNA sequence encoding modulators of TNF / NGF superfamily receptors, as well as protein, pharmaceutical composition, vector, methods and uses thereof. - Google Patents

Description

New proteins are described that bind to the intracellular regions of p55 and p75 TNF-R and Fas-it, which are capable of modulating the function of p55 and p75 TNF-R and Fas-R, and DNA sequences that code for them. New soluble oligomeric TNF-R, oligomeric Fas-R and oligomeric receptors with a mixture of TNF-R and Fas-R are also described. Methods for the production and use of all of the above are also described.

The field of invention

The present invention is generally in the field of receptors belonging to the TNF/NGF superfamily of receptors and the control of their biological functions. The TNF/NGF superfamily of receptors includes receptors such as p55 and p75 tumor necrosis factor receptors (TNF-R) and FAS ligand receptors (also called FAS/APO1 or FAS-R and will hereinafter be called FAS-R) and others. More specifically, the present invention relates to novel proteins that bind to the intracellular regions (IC) of p55 and p75 TNF-R and Fas-R (these intracellular regions are designated p55IC, p75IC and Fas-IC, respectively) and whose novel proteins are able to modulate the function of p55 and p75 TNF-Rs and Fas-R. One of the proteins capable of binding p55IC by the intact p55-TNF-R is P55IC itself in the form of a p55IC molecule or a part thereof, such as for example the so-called "death region" (DD) of p55IC. The present invention also relates to new applications The present invention also relates to the production and use of these new p55 and p75 TNF-F-binding proteins and Fas-R binding proteins, referred to here as p55 IC-, p75IC- and Fas-IC-binding proteins.

Background for the invention

Tumor necrosis factor (TNF-a) and lymphotoxin (TNF-p) (hereafter TNF refers to both TNF-a and TNF-P) are multifunctional pro-inflammatory cytokines produced mainly by mononuclear phagocytes, which have many effects on cells (Wallach, D. ( 1986) in: Interferon 7 (Ion Gesser, ed.), pp. 83-122, Academic Press, London; and Beutler and Cerami (1987)). Both TNF-α and TNF-0 initiate their effects by binding to specific cell surface receptors. Some of the effects are probably beneficial for the organism: they can destroy, for example, tumor cells or virally infused cells and increase the antibacterial activity of granulocytes. In this way, TNF contributes to the defense of the organism against tumors and infectious agents contribute to recovery from damage. TNF can thus be used as an anti-tumor agent in which application it binds to its receptors on the surface of tumor cells and thereby initiates the events that lead to the death of the tumor cells. TNF can also be used as an anti-infective agent.

However, both TNF-α and TNF-P have deleterious effects. There is evidence that overproduction of TNF-α may play an important pathogenic role in various diseases. Thus, effects of TNF-α primarily on the vasculature are known to be a main cause of the symptoms of septic shock (Tracey et al., 1986). In some diseases, TNF can cause severe weight loss (cachexia) by suppressing the activity of adipocytes and by causing anorexia, and TNF-a was thus named cachetin. It was also described as a mediator of tissue damage in rheumatic diseases (Beutler and Cerami, 1987) and as a major mediator of the damage observed with implant versus host reactions (Piquet et al., 1987). In addition, TNF is known to be involved in the inflammation process and in many other diseases.

Two distinct, independently expressed, receptors, p55 and p75 TNF-Rs, which bind both TNF-α and TNF-β specifically, initiate and/or mediate the above-mentioned biological effects of TNF. These two receptors have structurally different intracellular regions, suggesting that they signal differently (See Hohmann et al., 1989; Engelmann et al., 1990: Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990; Nophar et al., 1990; Smith et al., 1990; and Heller et al., 1990). However, the cellular mechanisms, for example, the different proteins and possibly other factors involved in the intracellular signaling of p55 and p75 TNF-R, have not yet been found (as indicated above, new proteins have been described for the first time that are in able to bind p75IC and p55 IC). It is this intracellular signaling that usually occurs after the binding of the ligand, i.e. TNF (a or P) to the receptor that is responsible for the initiation of the cascade of reactions that ultimately results in the observed response of the cell to TNF.

Regarding the above-mentioned cytocidal effect of TNF, this effect in most cells studied so far is mainly triggered by p55 TNF-R. Antibodies against the extracellular region (ligand binding region) of p55 TNF-R can themselves trigger the cytocidal effect (see EP 412486) which correlates with the efficiency of the receptor cross-linking of antibodies, which is considered to be the first step in the generation of the intracellular signaling process. Furthermore, mutational studies (Brakebusch et al., 1992; Tartaglia et al., 1993) have shown that the biological function of the p55 TNF-R depends on the integrity of its intracellular domain, and consequently it has been proposed that the initiation of the intracellular signaling leading to to the cytocidal effect of TNF occurs as a result of the association of two or more intracellular regions of p55 TNF-R. Furthermore, TNF (a and B) acts as a homotrimer and as such has been proposed to induce intracellular signaling via the p55 TNF-R by means of its ability to bind to and cross-link receptor molecules, i.e. cause receptor aggregation. It is described below how p55IC and p55DD can self-associate and induce TNF-associated effects in cells in a ligand-dependent manner.

Another member of the TNF/NGF superfamily of receptors is the FAS receptor (FAS-R) which has also been called the Fas antigen, a cell surface protein expressed in various tissues and which shares homology with a number of cell surface receptors including TNF-R and NGF-R. FAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991) and appears to serve as a negative selector for autoreactive T cells, i.e. during maturation of T cells, FAS-R mediates the apoptotic death of T cells that recognize self antigens. It has also been found that mutation in the FAS-R gene ( Ipr ) causes a lymphoproliferative disorder in mice corresponding to the human autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The ligand for FAS-R appears to be a cell surface-associated molecule carried by, among other things, killer T cells (or cytotoxic T lymphocytes - CTL) and thus when such CTL contact cells that carry FAS-R, they are able to induce apoptotic cell death to FAS-R bearing cells. Furthermore, a monoclonal antibody has been prepared that is specific for FAS-R, this monoclonal antibody is able to induce apoptotic cell death in cells bearing FAS-R, including mouse cells transformed with cDNA encoding human FAS-R (Itoh et al ., 1991).

It has also been found that various other normal cells besides T lymphocytes express FAS-R on their surface and can be killed by triggering this receptor. Uncontrolled induction of such a killing process is suspected of contributing to tissue damage in certain diseases, for example destruction of liver cells in acute hepatitis. Accordingly, finding ways to withhold the cytotoxic activity of FAS-R may have therapeutic potential.

In contrast, as it has been found that certain malignant cells and HIV-infected cells carry FAS-R on their surface, antibodies against FAS-R or the FAS-R ligand can be caused to trigger FAS-R mediated cytotoxic effects in these cells and thereby provide a means of combating such malignant cells or HIV-infected cells (see Itoh et al., 1991). The discovery of yet another way to increase the cytotoxic activity of FAS-R may thus have therapeutic potential.

There has been a long-felt need to provide a method for modulating the cellular response to TNF (a or B) and the FAS-R ligand, for example in pathological situations as mentioned above, where TNF or the FAS-R ligand is overexpressed, it is desirable to inhibit TNF- or FAS-R ligand-induced cytocidal effects, while in other situations, for example wound healing applications, it is desirable to increase the TNF effect, or in the case of FAS-R it is desirable in tumor cells or HIV-infected cells to increase the FAS-R mediated effect.

A number of approaches have been made by the inventors (see for example European Applications No. 186833, EP 308378, EP 398327 and EP 412486) to regulate the harmful effects of TNF by inhibiting the binding of TNF to its receptors using anti-TNF antibodies or by using soluble TNF receptors (which are mainly on the soluble extracellular regions of the receptors) to compete with the binding of TNF to the cell surface bound TNT-R. Furthermore, on the basis that the TNF binding to its receptors is necessary for the TNF-induced cellular effects, the inventors have made approaches (see for example EPO 568925) to modulate the TNF effect by modulating the activity of TNF-R. Briefly, EPO 568925 relates to a method for modulating signal transduction and/or cleavage in TNF-R whereby peptides or other molecules can interact either with the receptor itself or with effector proteins that interact with the receptor, thus modulating the normal function of TNF-R. In EPO 568925, the constructions and the characterization of different mutant p55 TNF-R, which have mutations in the extracellular, transmembrane and intracellular regions of p55 TNF-R, are described. In this way, regions within the above-mentioned regions of the p55 TNF-R are identified to be essential for the function of the receptor, i.e. the binding of the ligand (TNF) and the subsequent signal transduction and intracellular signaling that ultimately resulted in the observed TNF effect on the cells . Furthermore, a number of approaches have also been described to isolate and identify proteins, peptides or other factors capable of binding to different regions in the above-mentioned regions of TNF-R, whose proteins, peptides and other factors may be involved in the regulation or the modulation of the activity of TNF-R. A number of approaches for the isolation and cloning of DNA sequences encoding such proteins and peptides; for the construction of expression vectors for the production of these proteins and peptides, and for the production of antibodies or fragments thereof that interact with TNF-R, or with the above proteins and peptides that bind different regions of TNF-R, are also indicated in EPO 568925. However, no description is made in EPO 568925 of the actual proteins and peptides that bind to the intracellular regions of TNF-R (eg p55 TNF-R), nor is there any description of the yeast two-hybrid approach to isolate and identifying such proteins or peptides that bind to the intracellular regions of TNF-R. Correspondingly, no description of proteins or peptides capable of binding to the intracellular region of FAS-R has so far been made.

JBC, Vol. 267, No. 7, pp. 4304-4307, 1992, suggests that aggregation of the p55 TNF receptor intracellular domains, in the presence of a ligand, is an important component of the cellular toxicity signal. However, it is not suggested that the intracellular domains bind in the absence of ligand.

EP 0510691 discloses DNA coding for FAS. PNAS, Vol. 88, pp. 2830-2834, 1991, reports the cloning and expression of cDNA of two distinct murine TNF receptors and demonstrates that one receptor is species specific.

Thus, when it is desired to inhibit the effect of the TNF or FAS-R ligand, it will be desirable to reduce the amount or activity of TNF-R or FAS-R on the cell surface, while an increase in the amount or activity of TNF-R or FAS -R will be desirable when an increased TNF or FAS-R ligand effect is sought. In this regard, the promoters of both p55 TNF-R and p75 TNF-R have recently been sequenced and analyzed by the present inventors and a number of key sequence motifs have been found which are specific for various transcriptional regulatory factors and as such expression of these TNF-Rs can be controlled at the promoter level, i.e. inhibition of transcription from the promoters for a decrease in the number of receptors and an increase in transcription from the promoters for an increase in the number of receptors (see IL 104355 and IL 109633 and their corresponding, as yet unpublished EP and PCT counterparts). Corresponding studies regarding the control of FAS-R at the level of the promoter of the FAS-R gene have not yet been reported.

Furthermore, it must also be mentioned that while it is known that the tumor necrosis factor (TNF) receptors and the structurally-related receptor FAS-R, trigger in cells after stimulation by leukocyte-produced ligands, destwktive activities that lead to their own death, the mechanism of this release is still poorly understood. Mutational studies indicate that in FAS-R and the p55 TNF receptor (p55-R), signaling for cytotoxic activity involves distinct regions in their intracellular regions (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993). These regions ("dead regions") have sequence similarities. The "dead zones" of both FAS-R and p55-R tend to self-associate. Their self-association apparently promotes the receptor aggregation necessary for the initiation of signaling (as noted here below as well as by Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995) and at high levels of receptor expression, this can result in the triggering of ligand-independent signaling (as described below and by Boldin et al., 1995).

Thus, prior to the present invention, no proteins have been obtained that can deregulate the effect of the ligands belonging to the TNF/NGF superfamily, such as TNF or the FAS-R ligand effect on cells by mediating an intracellular signaling process, which signaling is probably controlled to a large extent by the intracellular domains (IC) of receptors belonging to the TNF/NGF superfamily of receptors such as those of TNF-R, i.e. p55 and p75 TNF-R intracellular domains (p55IC and p75IC, respectively) as well as FAS-IC.

Accordingly, it is an aim of the invention to provide proteins capable of binding to the intracellular regions of TNF-R and FAS-R, whose proteins are currently believed to be involved in the intracellular signaling process initiated by the binding of TNF to its receptors, or the binding of the FAS ligand to its receptor.

Another aim according to the invention is to provide antagonists (for example antibodies) for these intracellular region-binding proteins (IC-binding proteins) which can be used to inhibit the signaling process when desired, when such IC-bound proteins are positive signaling effects (i.e. induce signalling) , or to increase the signaling process when desired, when such IC-binding proteins are negative signaling effectors (ie, inhibit signaling).

Another aim of the invention is the use of such IC-binding proteins to isolate and characterize further proteins or factors that may for example be involved further downstream in the signaling process, and/or isolate or identify other receptors further upstream in the signaling process to which these IC-binding the proteins bind (for example other TNF-R or related receptors) and thus in whose function the IC-binding proteins are also involved.

Furthermore, an aim of the present invention is directed towards the use of the above-mentioned IC-binding proteins as antigens for the production of polyclonal and/or monoclonal antibodies. The antibodies can again be used for purification of the new IC-binding proteins from different sources, such as cell extracts or transformed cell lines.

Furthermore, these antibodies can be used for diagnostic purposes, for example for the identification of disorders related to abnormal function of cellular effects mediated by the receptors belonging to the TNF/NGF receptor superfamily.

A further aim of the invention is to provide pharmaceutical compositions comprising the IC-binding proteins above and pharmaceutical compositions comprising the antagonists for the IC-binding proteins, for the treatment or prophylaxis of TNF-induced or FAS ligand-induced conditions, for example such compositions can be used to increasing the TNF or FAS ligand effect or inhibiting the TNF or FAS ligand effect depending on the above-mentioned nature of the IC-binding protein or antagonist thereof contained in the composition.

Furthermore, according to yet another object of the invention, other ways of eliminating or antagonizing endogenously formed or exogenously administered TNF or FAS-R ligand are described using soluble oligomeric TNF-R, oligomeric FAS-R or oligomers which are a mixture of TNF -R and FASS-R. In this regard, it may be mentioned that an attempt in this direction was the isolation and recombinant production of a TNF-binding protein called TBP-I which was shown to be able to antagonize the effects of TNF. This antagonism was determined by both measuring the reduction of the cytotoxic activity of TNF as well as measuring the interference of TNF binding to its receptors (EP 308 378). TBP-I was shown to protect cells from TNF toxicity at concentrations of a few nanograms per ml and to interfere with the binding of both TNF-a and TNF-fi to cells, when given simultaneously with these cytokines. Further investigation of the mechanism by which TBP-I works revealed that TBP-I does not interact with the target cell, but rather blocks the function of TNF by specifically binding TNF, thus competing with TNF for the TNF receptor.

Accordingly, with another purification technique, the presence of two active components was found: one, TBP-I, and a second TNF-binding protein called TBP-II (first described in EP 398327). Both proteins provide protection against the in vitro cytocidal effect of TNF and both bind TNF-B less efficiently than TNF-a. Although according to SDS PAGE analysis the two proteins, TBP-I and TBP-II, appear to have a very similar molecular weight, they can be clearly separated from each other by a lack of immunological cross-reactivity, different N-terminal amino acid sequences and different armnoic acid composition.

However, the aforementioned soluble TNF binding proteins are monomeric and are able to bind only one monomer of the TNF homotrimer, the natural ligand that still allows TNF activity (ie incomplete neutralization) of TNF even two active monomers unbound to TNF binding proteins. Furthermore, soluble FAS-R (soluble FAS-R ligand-binding proteins) capable of binding FAS-R ligand, which is known to be a homotrimeric, cell surface-associated molecule, has not been described to date.

A so-called "dead zone" of p55-IC (Tartaglia et al., 1993) has been described, but did not show, as in the present invention, that p55-IC and the "dead zone" of this self-associate, where this self-association is primarily responsible for the signaling that leads to the induction of cell cytotoxicity. Furthermore, this publication is silent on the possibility of production of soluble, oligomeric TNF-R, or soluble, oligomeric Fas-R, or mixed oligomers thereof, moreover, it does not describe other TNF-related effects induced by p55-IC or parts thereof, e.g. EL-8 gene expression induction, all of which are described in the present invention. Likewise, another publication, published after the priority date of the present invention, describes the aggregation (i.e., self-association) ability of p55-IC, but does not, as noted above, relate to the use thereof to produce soluble, oligomeric TNF-R or Fas- R or other TNF-associated effects induced in a ligand-independent manner by p55-IC or parts thereof according to the invention.

Summary of the invention

According to the present invention, new proteins have been found which are capable of binding to either the intracellular region of p55 NTF-R (the p55IC-binding proteins) of p75 NTF-R (the p75IC-binding proteins) and FAS-R (the FAS -the IC-binding proteins). These p55IC, p75IC and FAS-IC binding proteins may act as mediators or modulators of TNF or FAS-R ligand activity on cells by mediating or modulating intracellular signaling processes that normally occur after binding of TNF to p55 and/or p75 TNF-R, or the binding of FAS-R ligand to the cell surface. Furthermore, it has been surprisingly and unexpectedly found that p55IC and FAS-IC are capable of self-association and that the fragments of p55IC and FAS-IC are similarly capable of binding to p55 IC, especially the so-called "death domains" (DD) in the IC of these receptors, i.e. p55DD and FAS-DD. Thus, p55 IC and FAS-IC and their fragments also represent proteins capable of binding to p55IC and FAS-IC and can thus be modulators of TNF or FAS-R ligand activity on cells.

Furthermore, the nature of the binding of one of the new proteins according to the invention, hereafter designated 55.11 protein, to the intracellular region of p55-TNF-R, has been more fully explained (see example 1).

Furthermore, according to another aspect, the present invention is based on the finding that the intracellular region of the p55 TNF receptor (p55-IC), a region contained therein, the so-called p55-IC "death region11, the intracellular region of the Fas/APOI receptor ( Fas-IC), and a region contained therein, the so-called Fas-IC "death region" is capable of self-association. Accordingly, it is possible to construct by standard recombinant DNA techniques a soluble oligomeric TNF receptor which is a fusion product containing at least two extracellular regions of a TNF receptor at one end thereof, and at the other end at least two of the above self-associating intracellular regions or portions thereof that self-associate to give an oligomer having at least two such fusion products bound together. Such soluble, oligomeric TNF-R is thus capable of binding two monomers of naturally occurring TNF homotrimers, and as such is effective in neutralizing TNF activity. tivity is desirable in all of the above-mentioned conditions where TNF is overproduced endogenously or is administered exogenously in high doses to result in unwanted side effects. Furthermore, the effective binding of TNF by soluble oligomeric receptors according to the invention may also serve to allow binding of exogenously added TNF and its subsequent desired slow release in conditions where TNF is administered for its beneficial effects, for example in tumor therapy. Likewise, it is also possible to construct standard recombinant DNA techniques an oligomeric FAS-R which is a fusion product containing at least two extracellular regions of a FAS-R at its one end and at its other end at least two of the above-mentioned self-associated intracellular regions or portions thereof that self-associate to give an oligomer having at least two such fusion products bound together. Such an oligomeric FAS-R is thus capable of binding two monomers of naturally occurring FAS-R ligand homotrimers, and as such effectively neutralizes FAS-R ligand activity. The neutralization of the FAS-R hgand activity is desirable in all of the above-mentioned conditions where excess amounts thereof are associated with undesirable side effects. In a similar way and in light of recent reports that indicate a possible association between TNF and FAS-R ligand-induced effects on cells and thus also a possible association, geographically on the cell surface where they bind to their receptors, it is also possible to construct by standard recombinant DNA techniques a mixed oligomeric receptor that has specificity for both TNF and FAS-R ligand. Such a mixed oligomer will be a mixture of the above-mentioned fusion products containing at least one extracellular region of a TNF-R and at least one extracellular region of a FAS-R at its one end, and at its other end at least two of the aforementioned themselves -associating intracellular regions or portions thereof which self-associate to give a mixed oligomer having at least two such fusion products bound together. Such a mixed oligomer is thus capable of binding at least one monomer of TNF and one monomer of FAS-R ligand at the same time to thereby reduce or effectively neutralize the TNF and FAS-R ligand activities at the cell surface in conditions as stated above where excess amounts of these two the cytokines are associated with the unwanted cellular effects. As noted above, the FAS-R ligand is usually cell surface-associated and recent reports also describe cell surface-associated forms of TNF. These mixed TNF-R/FAS-R oligomers are thus particularly useful for neutralizing TNF and FAS-R ligand activities on the cell surface.

Consequently, the present invention provides a DNA sequence that codes for a protein capable of binding one or more of the intracellular regions of one or more receptors belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) superfamily of receptors.

In particular, the present invention provides a DNA sequence selected from the group comprising: (a) a cDNA sequence derived from the coding region of a native TNF-R

intracellular domain binding protein;

(b) DNA sequences osm are capable of hybridization to a DNA of (a) under moderately stringent conditions and which encodes a biologically active TNF-R intracellularly

domain-binding protein, and

(c) DNA sequences which are degenerate as a result of the genetic code of the DNA sequences defined in (a) and (b) and which encode a biologically active TNF-R intracellular domain binding protein.

The present invention also provides a DNA sequence selected from the group comprising: (a) a cDNA sequence derived from the coding region of a native FAS-R

intracellular domain binding protein;

(b) DNA sequences capable of hybridization to a cDNA of (a) under moderately stringent conditions and which encode a biologically active FAS-R

intracellular domain binding protein; and

(c) DNA sequences which are degenerate as a result of the genetic code of DNA sequences defined in (a) and (b) and which encode a biologically active FAS-R intracellular domain binding protein.

In embodiments of the present invention, the DNA sequences encode p55 TNF-R, p75 TNF-R and FAS-R intracellular domain binding proteins, such as those encoding the herein designated proteins 55.1, 55.3, 55.11, 75.3, 75.16, F2, F9 and DD11. The present invention also provides a protein or analogues or derivatives thereof which are encoded by any of the above-mentioned sequences according to the invention, where said proteins, analogues and derivatives are capable of binding to one or more of the intracellular regions of one or more TNF-R or FAS-R. Embodiments of this aspect of the invention include the herein designated proteins 55.1, 55.3, 55.11, 75.3, 75.16, F2, F9 and DD11, their analogs and their derivatives.

The present invention also provides vectors that code for the above-mentioned proteins according to the invention which contain the DNA sequences above according to the invention, where these vectors are capable of being expressed in suitable eukaryotic or prokaryotic host cells; transformed eukaryotic or prokaryotic cells containing such vectors, and a method for producing such proteins, analogues or derivatives according to the invention by culturing such transformed host cells under conditions suitable for the expression of said protein, analogues or derivatives, carrying out post-translational modification of said protein as necessary for obtaining said protein and extracting said expressed protein analogues or derivatives from the cellular medium of said transformed cells or from cell extracts of said transformed cells.

The invention also enables antibodies or active derivatives or fragments thereof which are specific for the proteins, analogues or derivatives thereof according to the invention.

The invention also enables various applications of the above-mentioned DNA sequences or the proteins they encode according to the invention, whose applications include among others: (i) a method for modulating TNF or FAS-R ligand effect on cells carrying a TNF-R or a FAS -R, comprehensive treatment of said cells with one or more proteins, analogues or derivatives selected from the group comprising proteins, analogues and derivatives according to the invention, and a protein which is p55IC, p55DD, FAS-IC or FAS-DD analogues or derivatives thereof, all of which proteins are capable of binding to the intracellular area and modulating the activity of said TNF-R or FAS-R, wherein said treatment of the cells comprises introducing into said cells said one or more proteins, analogues or derivatives in a form suitable for intracellular administration or introduction into said cells in the form of a suitable expression vector, the DNA sequence that codes for said one or more proteins ner, analogues or derivatives; (ii) a method for modulating TNF or FAS-R ligand effect on cells carrying a TNF-R or a FAS-R comprising treatment of said cells with antibodies or active derivatives or fragments thereof according to the invention; (iii) a method for modulating the TNF or FAS-R ligand effect on cells carrying a TNF-R or FAS-R comprising treating said cells with an oligonucleotide sequence that codes for an antisense sequence of at least part of the sequence according to the invention, or codes for an antisequence of the p55IC, p55DD, FAS-IC or FAS-DD sequence, where said oligonucleotide sequence is able to block expression of at least one of the TNF-R or FAS-R intracellular domain binding proteins; (iv) a method for modulating the TNF or FAS-R ligand activity on cells bearing a TNF-R or FAS-R comprising: (a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein that is in capable of binding to a specific cell surface receptor and a sequence selected from among an oligonucleotide sequence encoding an antisense sequence of at least part of the sequence according to the invention and an oligonucleotide sequence encoding an antisense sequence of p55IC, p55DD, FAS-IC or FAS- the DD sequence, where said oligonucleotide sequence is capable of blocking the expression of at least one of TNF-R or FAS-R intracellular domain-binding proteins when it is introduced into said cells by said virus; and

(b) infecting said cells with said vector of (a);

(v) a method of modulating the TNF or FAS-R ligand effect on cells bearing TNF-R or FAS-R, comprising treating said cells with a suitable vector encoding a ribozyme having a sequence specific for a sequence selected from an mRNA sequence that codes for a protein, an analog or a derivative according to the invention and an mRNA sequence that codes for a protein, an analog or a derivative thereof according to the invention and an mRNA sequence that codes for p5IC, p55DD, FAS-IC or FAS -DD, where said ribozyme sequence is capable of interacting with said mRNA sequence and capable of cleaving said mRNA sequence to result in inhibition of the expression of the protein, analog or derivative according to origin or of the expression of p55IC, p55DD, FAS- IC or FAS-DD,

(vi) a method for treating tumor cells or HIV-infected cells or other diseased cells, comprising: (a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein capable of binding to a tumor cell surface receptor or HIV -infected cell surface receptor or capable of binding to another cell surface receptor on other diseased cells and a sequence selected from among a sequence according to the invention which codes for a protein, an analog or a derivative according to the invention and a sequence which codes for p55IC, p55DD, FAS -IC, FAS-DD or a biologically active analogue or derivative thereof, where said protein, analogue or derivative according to the invention, p55IC, p55DD, FAS-IC, FAS-DD, analogue or derivative, when expressed in said tumor cell or HIV-infected cell or other diseased cell, capable of killing said cell; and (b) infecting said tumor cells or HIV-infected cells or other infected cells with said vector according to (a), (vii) a method for isolating and identifying proteins, factors or receptors capable of binding to the intracellular domain-binding proteins according to the invention, comprehensive application of the procedure for affinity chromatography where said protein according to the invention is connected to the affinity chromatographic matrix, said connected protein is brought into contact with a cell extract and proteins, factors or receptors from the cell extract that bind to said connected protein are then eluted, isolated and analyzed; (viii) a method for isolating and identifying proteins capable of binding to the intracellular domain-binding proteins according to the invention, comprising the use of the yeast two-hybrid procedure where a sequence coding for said intracellular domain-binding protein is carried out by a hybrid vector and a sequence from a cDNA or genomic DNA library is carried out by the second hybrid vector, the vectors are then used to transform yeast host cells and the positive transformed cells are isolated, followed by extraction of said second hybrid vector to obtain a sequence encoding a protein which binds to said intracellular domain binding protein; and

(ix) a method for isolating and identifying a protein capable of

bind to the intracellular regions of TNF-R or FAS-R comprising application of the procedure of non-stringent southern hybridization followed by PCR cloning, in which a sequence or part thereof according to the invention is used-as a probe to bind sequences from a cDNA or genomic DNA library that has at least partial homology to this, where said bound sequences are then amplified and cloned by the PCR procedure to give clones that code for proteins that have at least partial homology to said sequences according to the invention.

The present invention also provides a pharmaceutical composition for modulating the TNF or FAS ligand effect on cells comprising, as active ingredient, any of the following: (i) a protein according to the invention, or the protein p55IC, p55DD, FAS-IC or FAS-DD, its biologically active fragments, analogues, derivatives or mixtures thereof; (ii) a recombinant animal virus vector encoding a viral surface protein capable of binding to a TNF-R or FAS-R bearing cell, or tumor cell-specific receptor, and a sequence encoding a protein, an analog or a derivative according to invention or codes for p55IC, p55ID, FAS-IC or FAS-DD; (iii) a recombinant animal virus vector encoding a viral surface protein as in (ii) above and an oligonucleotide sequence encoding an antisense sequence of p55IC, p55ID, FAS-IC or FAS-DD sequence; and (iv) a vector encoding a ribozyme of sequence capable of interacting with an mRNA sequence encoding a protein, an analog or a derivative according to the invention or an mRNA sequence encoding p55IC, p55DD, FAS-IC or

FAS-DD.

The invention thus enables the use of p55IC or DNA encoding it. This is based on the discovery that a p55-IC can in a ligand (TNF)-independent manner induce other TNF-associated effects in cells. Accordingly, a method for inducing TNF-associated effects in cells or tissues is enabled, comprising treating said cells with one or more proteins, analogues or derivatives thereof, wherein said one or more proteins are selected from a protein which is essentially the entire self-associating intracellular the region of p55 TNF-R (p55-IC) or portions thereof which are capable of self-associating and inducing, in a ligand (TNF)-independent manner, said TNF effect in the cells, where said treatment of the cells comprises introduction into said cells of one or more proteins, analogues or derivatives in a form suitable for intracellular introduction thereof, or introduction into said cells of a DNA sequence that codes for said one or more proteins, analogues or derivatives thereof in the form of a suitable vector carrying said sequence, where said vector is able to effect insertion of said sequence into said cells in a way that causes said sequence to be expressed in the cells.

Embodiments of the above method include:

(i) a method wherein said treatment of cells is by transfection of said cells with a recombinant animal viral vector comprising the steps of: (a) constructing a recombinant animal viral vector carrying a sequence encoding a viral surface protein (ligand) capable of to bind to a specific cell surface receptor on the surface of said cell to be treated, and a second sequence encoding a protein which is p55-IC, parts thereof, analogues or derivatives of all the foregoing, wherein said protein is expressed in said cells are capable of self-associating and inducing said one or more TNF-associated effects; and

(b) infecting said cells with the vector from (a);

(ii) a method where said TNF effect to be induced in said cells is the induction of BL-8 gene expression, where said vector carries a sequence which encodes mainly the entire p55-IC, parts thereof, analogues or derivatives of all the preceding which is capable, when expressed in the cells, of self-association and signaling for the induction of said IL-8 gene expression, (iii) a method for treating tumor cells or virally infected cells, or for weakening the antibacterial effect of granulocytes wherein said viral vector carries a sequence that codes for a viral ligand capable of binding a specific cell surface receptor on the surface of said tumor cells, virally infected cells or granulocytes and a sequence that codes for said p55-IC, parts thereof, analogs and derivatives thereof, which when expressed in said tumor, virally infected or granulocyte cells induces TNF-associated effects leading to the death of these cells, (iv) a method of b eaction of tumor cells wherein said p55-IC, parts thereof, analogues or derivatives thereof, when expressed in tumor cells, induces expression of IL-8 leading to the killing of said tumor cells by its chemotactic activity which attracts granulocytes and other lymphocytes to the tumor cells resulting in death of the tumor cells.

There is thus provided the intracellular region of p55-R (p55-IC), parts, analogs and derivatives of all the foregoing for use in treating cells by inducing therein TNF-associated effects; and the following embodiments thereof: (i) p55-IC, parts, analogues and derivatives for use in the treatment of cells by induction therein of IL-8 gene expression, (ii) p55-IC, parts, analogues and derivatives for use in the treatment of tumor cells by induction therein of IL-8 gene expression which results in the killing of the tumor cells.

Furthermore, there is provided a pharmaceutical composition for the treatment of cells by inducing therein TNF-associated effects, comprising, as active ingredient, p55-IC, parts thereof, analogs and derivatives of all the foregoing, and a pharmaceutically acceptable carrier; and the following embodiments thereof: (i) a pharmaceutical composition for the treatment of cells by inducing therein TNF-associated effects comprising as active ingredient a recombinant animal virus vector encoding p55-IC, parts thereof, analogs and derivatives thereof, and a protein which is able to bind a cell surface protein on the cells to be treated, (ii) a pharmaceutical composition for the treatment of tumor cells, where administration of said composition leads to induction of IL-8 expression, and subsequent killing of the tumor cells.

The present invention also enables a soluble, oligomeric tumor necrosis factor receptor (TNF-R) comprising at least two self-associating fusion proteins, where each fusion protein has (a) at one end a TNF-binding region selected from the extracellular region of a TNF-R, analogues or derivatives thereof, wherein said extracellular region, analogues or derivatives are incapable of harmful self-association and which are capable of binding TNF; and (b) at its other end, a self-associating region selected from (i) substantially the entire intracellular region of p55 TNF-R (p55-IC), running from about amino acid residue 206 to about amino acid residue 426 of the native p55 TNF -R molecule (p55-R); (ii) the dead region of p55-IC running from about amino acid residue 328 to about amino acid residue 426 of the native p55-R; (iii) essentially the entire intracellular region of the Fas/APO1 receptor (Fas-IC); (iv) the dead region of Fas-IC; and (v) analogs, fractions or derivatives of any of (i)-(iv) capable of self-association, wherein said two self-associated proteins self-associate only at the ends (b) having said ends (a) which is capable of binding to at least two TNF monomers, where each end (a) is capable of binding to one TNF monomer; and salts and functional derivatives of said soluble, oligomeric TNF-R.

Variants of said soluble TNF-R include all the aforementioned combinations of ends (a) with ends (b) as defined above, for example, a soluble, oligomeric TNF-R comprising as extracellular region, the p5S-R extracellular region and as self-associating intracellular region, pSS-IC.

Furthermore, a method for the production of soluble oligomeric TNF-R is conceivable comprising: (a) the construction of an expression vector encoding any one of said fusion proteins, where the DNA sequence of said ends of the fusion protein is obtained four cloned DNA sequences encoding mainly for the entire extracellular region of TNF-R, analogs or derivatives thereof; and from cloned DNA sequences encoding substantially all of said p55-IC, p55-IC dead region, Fas-IC, Fas-IC dead region, analogs or derivatives of all the preceding ends which are ligated together to form a fusion protein sequence, and said fusion protein sequence is inserted into said vector under the control of the transcriptional and translational regulatory sequences; (b) introducing the vector of (a) into a suitable host cell in which said fusion protein is expressed; and (c) purifying the fusion protein expressed in said host cell, wherein said fusion protein is self-associating before, during or after the purification process to yield a soluble, oligomeric TNF-R.

Furthermore, a vector is conceivable which codes for the above fusion proteins, which are useful in the methods according to the invention; host cells containing the vector as well as a pharmaceutical composition comprising the soluble, oligomeric TNF-R, salts or functional derivatives thereof and mixtures of any of the foregoing according to the present invention, as an active ingredient, together with a pharmaceutically acceptable carrier. Correspondingly, soluble, oligomeric TNF-R salts, functional derivatives thereof and mixtures of any of the foregoing are provided according to the invention, for use in antagonizing the harmful effects of TNF in mammals, especially in the treatment of conditions where an excess of TNF is formed endogenously or exogenously administered; or alternatively, for use in maintaining prolonged beneficial effects of TNF in mammals when used with exogenously administered TNF.

Along the lines indicated regarding the above aspect, it has been discovered that it is possible to construct a soluble, oligomeric Fas/APO1 receptor (Fas-R) useful for antagonizing the deleterious effects of the Fas ligand. Accordingly, a soluble, oligomeric Fas/APO1 receptor (Fas-R) is conceivable comprising at least two self-associated fusion proteins, each fusion protein having (a) at one end a Fas ligand-binding region selected from the extracellular region of a Fas-R , analogues or derivatives thereof which are not capable of self-association and which are capable of binding the Fas ligand; and (b) at its other end a self-associating region selected from (i) substantially the entire intracellular region of p55 TNF-R (p55-IC) running from about amino acid residue 206 to about amino acid residue 426 of the native p55 TNF-R molecule (p55-R); (ii) the dead region of p55-IC running from about amino acid residue 328 to about amino acid residue 426 in the native p55-R; (iii) essentially the entire intracellular region of the Fas/APO1 receptor (Fas-IC); (iv) the dead region of Fas-IC; and (v) analogs or derivatives of any of (i)-(iv) capable of self-association, wherein said at least two self-associating proteins only self-associate at said ends (b) having ends (a) capable of binding at least two Fas ligand monomers, each end (a) capable of binding one Fas ligand monomer; and salts and functional derivatives of said soluble, oligomeric Fas-R.

There is also provided a method for producing the soluble, oligomeric Fas-R comprising: (a) construction of an expression vector encoding any of said fusion proteins, wherein the DNA sequence of each of said ends of the fusion protein is obtained from cloned DNA sequences encoding substantially all of said extracellular region of Fas-R, analogs or derivatives thereof, and from cloned DNA sequences encoding substantially all of said p55-IC, p55-IC death region, Fas-IC, Fas-IC death region, analogs or derivatives thereof of all the foregoing, wherein said ends are ligated together to form a fusion protein sequence, and said fusion protein sequence is inserted into said vector under the control of the transcriptional and translational regulatory sequences; (b) introducing the vector (a) into a suitable host cell in which said fusion protein is expressed; and (c) purifying the fusion protein expressed in host cells, wherein said fusion protein self-associates before, during or after the purification process to yield a soluble, oligomeric Fas-R.

In addition, an expression vector containing the fusion protein sequence encoding the soluble, oligomeric Fas-R useful in the above method is also provided; host cells containing the vector and pharmaceutical compositions comprising the soluble, oligomeric Fas-R, salts or functional derivatives thereof, or mixtures of any of the foregoing as an active ingredient together with a pharmaceutically acceptable carrier. Similarly, there is provided a soluble, oligomeric Fas-R, salts or functional derivatives thereof or mixtures of any of the foregoing for use in antagonizing the deleterious effects of Fas ligand in mammals, particularly in the treatment of conditions in which an excess of Fas ligand is formed endogenously or is administered exogenously.

In a similar way as stated above regarding the oligomeric TNF-R and oligomeric FAS-R, it is also possible to prepare mixed oligomers which have binding specificity for both TNF and FAS-R ligand. Thus, the present invention also provides a mixed oligomeric TNF-R/FAS-R comprising at least two self-associated fusion proteins, one of which fusion proteins is selected from any of the above-mentioned TNF-specific fusion proteins, and the other fusion protein is selected from any of the above FAS-R ligand-specific fusion proteins to provide a mixed oligomer having at least one TNF-R extracellular region and at least one FAS-R extracellular region linked by self-association between the intracellular regions or portions hence fused to each of the extracellular areas. These mixed oligomeric receptors are produced by producing, as indicated above, oligomeric TNF-R and oligomeric FAS-R and then mixing these together and subsequent selection by standard procedures of the oligomers that have binding specificity for both FAS-R ligand and TNF. Another way of producing mixed oligomeric receptors is by cotransfecting appropriate host cells with vectors, as indicated above, which encode any of the TNF-specific fusion proteins (soluble TNF-R) and which encode any of FAS-R ligand-specific fusion proteins (soluble FAS-R), purification of the expressed fusion proteins that self-associate before, during, or after purification to yield oligomeric receptors, and then selection by standard procedures of the oligomeric receptors capable of binding to both TNF and FAS-R ligand.

Likewise, pharmaceutical compositions comprising mixed oligomeric receptors, salts or functional derivatives thereof or mixtures of any of the foregoing as active ingredient together with a pharmaceutically acceptable carrier are also provided. Additionally, mixed oligomeric receptors, salts or functional derivatives thereof or mixtures of any of the foregoing are provided for use in antagonizing the deleterious effects of both TNF and FAS-R ligands in mammals, particularly in the treatment of conditions where a excess TNF and FAS-R ligand are generated endogenously or are administered exogenously; or alternatively for use in maintaining prolonged (slow release) beneficial effects of TNF and/or FAS-R ligand in mammals when used with TNF and/or FAS-R ligand (in soluble form) administered exogenously.

The present invention comprises a DNA sequence that codes for a protein capable of binding to an intracellular domain of a receptor belonging to

the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor superfamily, preferably selected from the group consisting of p55 TNF-R, p75 TNF-R and the FAS ligand receptor, said DNA sequence being selected from the group consisting of:

(a) DNA sequences comprising a nucleotide sequence encoding the protein 55.1 having the amino acid sequence corresponding to amino acid residue 328 to residue 426 of the p55 TNF-R amino acid sequence; (b) DNA sequences comprising a nucleotide sequence encoding proE6n 55.3 with the amino acid sequence corresponding to amino acid residue 277 to residue 426 of the p55 TNF-R amino acid sequence; (c) DNA sequences comprising a nucleotide sequence encoding the protein 55.11 having the amino acid sequence given in Figure 4; (d) DNA sequences encoding a protein selected from the group consisting of the herein designated proteins 75.3 and 75.16 comprising the nucleotide sequence given in Figure 1(b) and Figure 1(c), respectively; (e) DNA sequences encoding any of the proteins F2, F9 and DD11, comprising a DNA sequence selected from the group consisting of the nucleotide sequence given in the first and second parts of Figure 10, respectively, the nucleotide sequence given in the first, second and third part of Figure 11 and the nucleotide sequence given in Figure 12; (f) DNA sequences capable of hybridizing to a DNA sequence according to any of items (a) to (e) under moderately stringent conditions, and (g) DNA sequences which are degenerate as a result of the the genetic code relative to the DNA sequences according to any one of items (a) to (f).

The present invention also includes a vector comprising a DNA sequence according to claim 1.

Furthermore, the present invention comprises a transformed eukaryotic or prokaryotic host cell containing a vector according to claim 2.

Furthermore, the present invention comprises a method for the production of the protein, analogues or derivatives which are capable of binding to an intracellular domain of a receptor belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) - receptor superfamily, including cultivation of the transformed host cell according to requirements 3 under conditions suitable for expression of said protein, analogues or derivatives, causing post-translational modification of said protein as necessary for obtaining said protein and extraction of said expressed protein, analogues or derivatives from the culture medium of said transformed cell or from the cell extracts of said transformed cell .

Furthermore, the present invention comprises a protein, or analogues and derivatives thereof, characterized in that it is able to bind to an intracellular domain of a TNF or FAS receptor encoded by a DNA sequence according to claim 1 or produced according to the procedure in claim 4.

Furthermore, the present invention comprises the use of one or more proteins, analogues or derivatives selected from the group consisting of the proteins, analogues and derivatives according to claim 5 and a protein which is p55IC, p55DD, FAS-IC or FAS-DD, analogues or derivatives thereof , all said proteins being capable of binding to the intracellular domain and modulating the activity of said TNF-R or FAS-R, for the production of a pharmaceutical composition for modulating the TNF or FAS-R ligand effect on cells bearing a TNF -R or a FAS-R, where said pharmaceutical composition comprises said one or more proteins, analogues or derivatives in a form suitable for intracellular introduction into cells, or a DNA sequence that codes for said one or more proteins, analogues or derivatives in form of a suitable vector carrying said sequence, said vector being able to cause insertion of said sequence into said cells in such a way that said sequence moved in said cells.

Furthermore, the present invention comprises the use of an oligonucleotide sequence selected from a sequence that codes for an antisense sequence of at least part of the sequence according to claim 1, and a sequence that codes for the antisense sequence of p55IC, p55DD, FAS-IC or FAS-DD, wherein said oligonucleotide sequence is capable of blocking the expression of at least one of the TNF-R or FAS-R intracellular domain-binding proteins for the preparation of a pharmaceutical composition for modulating the TNF or FAS-R ligand action on cells bearing a TNF-R or a FAS-R.

Furthermore, the present invention comprises the use of a recombinant animal virus vector carrying a sequence encoding a viral surface protein capable of binding to a specific tumor cell surface receptor or HTV-infected cell surface receptor or receptor carried by other diseased cells and a sequence encoding for a protein, analog or derivative according to claim 5, which, when expressed in said tumor cell, HIV-infected cell or other diseased cell, is capable of killing said cell, for the preparation of a pharmaceutical composition for the treatment of tumor cells or HIV-infected cells or other diseased cells.

Furthermore, the present invention includes the use of one or more proteins,

analogues or derivatives according to claim 5, for the preparation of a pharmaceutical composition for the induction of TNF-associated effects in cells or tissues, where said pharmaceutical composition comprises said one or more proteins, analogues or derivatives in a form suitable for intracellular introduction into cells, or a DNA sequence which codes for said one or more proteins, analogues or derivatives in the form of a suitable vector carrying said sequence, said vector being able to cause insertion of said sequence into said cells in such a way that said sequence is expressed in said cells.

Furthermore, the present invention comprises the use of a vector that codes for a ribozyme sequence capable of interacting with a cellular mRNA sequence that codes for a protein according to claim 5 or an mRNA sequence that codes for p55IC, p55DD, FAS-IC or FAS -DD for the preparation of a pharmaceutical composition for modulating the TNF or FAS-R ligand effect on cells designed in a manner that allows expression of said ribozyme sequence in said cells, and when said ribozyme sequence is expressed in said cells it interacts with said cellular mRNA sequence and cutting said mRNA sequence, resulting in expression inhibition of said protein or said p55IC, p55DD, FAS-IC or FAS-DD in said cells.

Furthermore, the present invention comprises a method for isolating and identifying proteins capable of binding the intracellular domain-binding proteins according to claim 5, comprising the use of the yeast two-hybrid procedure where a sequence coding for said intracellular domain-binding protein is carried by one hybrid vector and sequence from a cDNA or genomic DNA library is carried by the second hybrid vector, the vectors are then used to transform yeast host cells and the positively transformed cells are isolated, followed by extraction of said second hybrid vector to obtain a sequence encoding a protein that binds to said intracellular domain binding protein.

Furthermore, the present invention comprises a pharmaceutical composition for modulating the TNF or FAS-R ligand effect on cells comprising a protein according to claim 5 or the protein p55IC, p55DD, FAS-IC or FAS-DD, its biologically active fragments, analogues, derivatives or mixtures thereof.

Furthermore, the present invention comprises a pharmaceutical composition for modulating the TNF or FAS-R ligand action on cells comprising a recombinant animal virus vector which codes for a protein capable of binding a cell surface receptor and which codes for a protein according to claim S, or the protein p55IC, p55DD, FAS-IC or FAS-DD, its biologically active fragments or analogues.

Furthermore, the present invention comprises a pharmaceutical composition for modulating the TNF or FAS-R ligand action on cells comprising an oligonucleotide sequence that codes for an antisense sequence according to the sequence according to claim 1.

Furthermore, the present invention comprises the use of a protein according to claim S for the production of a pharmaceutical composition, for the treatment of tumours.

Other aspects and embodiments of the present invention are also provided as will be apparent from the following detailed description of the invention.

It should be noted that the following expressions: "Modulation of the TNF effect on cells" and "Modulation of the FAS ligand effect on cells" throughout the specification are intended to include in vitro as well as in vivo treatment.

Brief description of the figures

Fig. 1a-c schematically indicate the partial and preliminary nucleotide sequence of the cDNA

clones encoding p55IC and p75IC-binding proteins, where fig. 1(a) is the sequence of clone 55.11 encoding the p55IC-binding protein 55.11; fig. 1(b) is the partial and preliminary sequence of clone 75.3 encoding p75IC-binding protein 75.3; and fig. 1(c) is the partial and preliminary sequence of clone 75.16 encoding p75IC-binding protein p75.16; all as described in Example 1; and fig. 1(d) indicates the deduced amino acid sequence of protein 55.11, derived from the nucleotide sequence of FIG. l(a), as also described in example 1.

Fig. 2 is a reproduction of a Northern blot showing the 55.11-specific mRNA

which is present in a number of the tested cell lines, as described in example 1.

Fig. 3A and B are reproductions of autoradiograms showing in vitro binding of the protein encoded by the 55.11 cDNA to GST fusion protein-containing portions of p55-IC, where in Fig. 3A the binding of the full-length 55.11 protein (55.11 full) to various GST fusion proteins is indicated, and in fig. 3B shows the binding of a portion of 55.11 linked to the FLAG octapeptide to various GST fusion proteins, all described in Example 1. Fig. 4 schematically shows a comparison of the deduced amino acid sequence of human 55.11 to related protein sequences derived from lower organisms, as described in example 1. Fig. 5 is a reproduction of a Western blot stained with anti-MBP polyclonal antiserum showing the self-association of p55IC, Western blot derived from an SDS-PAGE gel on which the interacting bacterially produced chimeric protein p55IC was electrophoresed -MBP and p55IC-GST (lanes 1-4) or the control interaction between the chimeric protein p55IC-MBP and GST alone (lanes 5-8), the interactions between the chimeric proteins (and control) performed on glutathione-agarose beads before SDS- PAGE, as described in Example 2. Fig. 6 is a reproduction of phase contrast micrographs showing the cytotoxic effect of full-length p55IC in HTta1 cells transfected with an exp recombinant vector encoding this p55IC (right panel); and the inhibition of the cytotoxic effect when the expression of the vector is blocked by treatment of the cells with tetracycline (left panel), as described in Example 2. Fig. 7 shows the ligand-independent release of the cytotoxic effect in HeLa cells transfected with full-length p55-R, its intracellular region, or parts of the intracellular region including the "dead region", where: (i) on the far left side of FIG. 7 shows schematically the different DNA molecules that code for full-length p55-R, its intracellular region and parts of the intracellular region that are inserted into the vector into which the HeLa cells were transfected; (ii) the left and middle bars show TNF receptor expression in HeLa cells of each of the types of receptor shown on the far left in fig. 7, the left bar represents the amounts of receptor in ng/cell sample and the middle bar represents the amounts of receptor expressed by radioiodinated TNF bound to the transfected cells, and (iii) the right bar shows the viability of the HeLa cells expressing the different types of receptor; and wherein all bar graphs with white bars represent cells transfected in the presence of tetracycline and shaded bars represent cells transfected in the absence of tetracycline; this is also described in example 2. Fig. 8 shows the ligand-independent induction of IL-8 gene expression in HeLa cells transfected with full-length p55-R or its intracellular region (p55IC), where in panel A a reproduction is shown of a Northern blot showing the Northern analysis of RNA extracted from HeLa cells treated or not treated with TNF (the two lanes on the left labeled "control" and "TNF"), and RNA extracted from HeLa cells transfected with vectors encoding for p55-R, p55-IC or the control protein, luciferase (the rest of the tracks represent "p55-IC", "p55-R" and Lue, respectively), where the cells that have been transfected in the presence (+) or absence (-) of tetracycline in each case (thus two lanes per transfection); and wherein in panel B there is shown the methylene blue staining of 18S rRNA in each of the HeLa cell samples shown in panel A; all of the above is described in example 2. Fig. 9 (A and B) graphically indicates the ligand-dependent release of a cytocidal effect in HeLa cells transfected with p55R or parts thereof, or with AS-IC, in which fig. 9A the results with regard to p55R or parts thereof are indicated, and in fig. 9B, the results with regard to FAS-IC are indicated. In the panels on the left in both Figs. 9A and B, the proportion of p55R or FAS-IC used in the transfections is indicated schematically, while the panels on the right graphically indicate the experimental results, all as described in example 2. Fig. 10 graphically shows the partial and preliminary nucleotide sequence of a cDNA

clone, named "F2", which encodes a protein capable of binding to p55IC and FAS-IC, as described in Example 3.

Fig. 11 graphically shows the partial and preliminary nucleotide sequence of a cDNA clone,

called F9, which encodes a protein capable of binding to p55IC and FAS-IC, as described in Example 3.

Fig. 12 schematically indicates the partial and preliminary nucleotide sequence of a cDNA

clone, named DD11, which encodes a protein capable of binding to p55IC, particularly p55DD, and FAS-IC, as described in Example 3.

A detailed description of the invention

The present invention relates, according to one aspect, to new proteins that are able to bind to the intracellular region of receptors belonging to the TNF/NGF superfamily, such as TNF-R and FAS-R and are thus considered mediators or modulators of this superfamily receptors, for example TNF-R and FAS-R, which have a role in, for example, the signaling process that is initiated by the binding of TNF to TNF and the FAS ligand to FAS-R. Examples of these proteins are those that bind to the intracellular region of p55 TNF-R (p55IC), such as the proteins designated herein as 55.1, 55.3 and 55.11 (Example 1) as well as those encoded by the cDNA clones F2, F9 and Ddl 1 (Example 3), those that bind to the intracellular domain v p75 TNF-R (p75IC), such as the proteins designated 75.3 and 75.16 (Example 1); and those that bind to the intracellular region of FAS-R (FAS-IC), such as the proteins encoded by cDNA clones F2, F9 and DD11 (Example 3). Proteins 55.1 and 55.3 have been found to represent parts or fragments of the intracellular domain of p55 TNF-R (p55IC); other proteins, 55.11,75.3 and 75.16, represent proteins that have not been described at all prior to the present invention (75.3,75.16) or those that have been described

(55.11, see Klan et al., 1992), but whose function and other characteristics, particularly the ability to bind to a TNF-R, were not described in any way (see Example 1, below). The new proteins encoded by the cDNA clones F2, F9 and DD11 also represent proteins that had not been described at all before, i.e. their sequence is not in the "GENEBANK" or "PROTEIN BANK" databases of DNA or amino acid sequences.

The present invention thus relates to DNA sequences which code for these proteins and the proteins encoded by these sequences.

Moreover, the present invention also relates to the DNA sequences which code for biologically active analogues and derivatives of these proteins and the analogues and derivatives encoded thereby. The preparation of such analogues and derivatives is by standard procedure (see for example Sambrook et al., 1989) in which the DNA sequences that code for these proteins, one or more codes can be deleted, added or substituted by others, to give analogues that has at least one amino acid residue change with respect to the native protein. Acceptable analogs are those that retain at least the capacity to bind to the intracellular region of the TNF/NGF receptor superfamily, such as FAS-R or TNF-R, such as p55IC, p75IC, or FAS-IC, or that can mediate any binding or enzymatic activity, for example analogs that bind p55, p75IC or FAS-IC, but do not signal, i.e. do not bind to a further downstream receptor, protein or other factor, or do not catalyze a signal-dependent reaction. In such a way, analogues can be produced which have a so-called dominant-negative effect, namely an analogue which is defective either in binding to, for example, p55IC, p75IC or FAS-IC, or in subsequent signaling after such binding. Such analogues can, for example, be used to inhibit the TNF or FAS ligand effect by competing with neutral IC-binding proteins. Likewise, so-called dominant-positive analogues can be produced which will serve to enhance, for example, the TNF or FAS ligand effect. These will have equal or better IC-binding properties and the same or better signaling properties as the natural IC-binding proteins. Similarly, derivatives can be prepared by standard modifications of the side groups on one or more amino acid residues of the proteins, or by conjugation of the proteins to the other molecule, for example an antibody, enzyme, receptor, etc., which are well known in the art.

The new TNF-R and FAS-R intracellular domain binding proteins, for example the proteins 55.1, 55.3, 55.11, 75.3,75.16 as well as the proteins encoded by the cDNA clones F2, F9 and DD11 (hereafter F2, F9 and DD11) have a number of possible applications , for example: (i) They can be used to mimic or increase the function of the TNF or FAS-R ligand in situations where an increased TNF or FAS-R ligand effect is desired such as in anti-tumor, anti-inflammatory or anti- HTV applications where TNF- or FAS-R ligand-induced cytotoxicity is desirable. In this case, the proteins, for example those that bind to p55IC such as 5.1, 55.3, as well as F2, F9 and DD11, and the free p55IC itself (see below and Example 2), as well as the "dead zone" of p55IC ( p55DD), which increases the TNF effect; or proteins F2, F9 and DD11 as well as FAS-IC and FAS-DD which increase the FAS-R ligand effect, i.e. cytotoxic activity, can be introduced to the cells by standard procedures known per se. For example, as the proteins are intracellular and it is

desirable that they are introduced only to the cells where the TNF or FAS-R ligand effect is desired, a system that is specific for the introduction of these proteins into the cell is necessary. One way of accomplishing this is by the creation of a recombinant animal virus, for example one derived from Vaccinia, into which DNA the following two genes will be introduced: the gene that codes for a ligand that binds to cell surface proteins that are specifically expressed by the cells, for

for example one such as the AIDs (HIV) virus gp 120 protein that binds specifically to some cells (CD4 lymphocytes and related white blood cells) or any other ligand that binds specifically to cells bearing a TNF-R or FAS-R, such that the recombinant viral vector will be able to bind such TNF-R or FAS-R bearing cells; and the gene encoding the novel intracellular domain binding protein or the p55IC, p55DD, FAS-IC or FAS-DD protein. Thus, expression of the cell surface binding protein on the surface of the virus will target the virus specifically to the tumor cell or other TNF-R or FAS-R bearing cell, after which the intracellular domain binding protein coding sequence or p55IC, p55DD, FAS-IC or FAS-DD coding the sequence will be introduced into the cells via the virus, and as soon as it is expressed in the cells, it will result in an increase in the TNF or FAS-R ligand effect which leads to the death of the tumor cells or other TNT-R or FAS-R-bearing cells that is desirable to euthanize. Construction of such an animal virus is done by standard procedures (see, for example, Sambrook et al., 1989). Another possibility is to introduce

the sequences of the new proteins or p55IC, p55DD, FAS-IC or FAS-DD in the form of oligonucleotides that can be absorbed by the cells and expressed there.

(ii) They can be used to inhibit the TNF or FAS-R ligand effect, for example in cases such as tissue damage during septic shock, graft-versus-host rejection, or acute hepatitis, in which case it is desirable to block TNF-induced TNF -R or FAS-R ligand-induced FAS-R intracellular signaling. In this situation, it is possible, for example, to introduce into the cells, by standard procedures, oligonucleotides that have an anti-sense coding sequence for these new proteins, or anti-sense coding sequence for p55IC, p55DD, FAS-IC or FAS-DD, which will effectively block the translation of mRNA encoding these proteins and thereby block their expression and lead to inhibition of TNF- or FAS-R

the ligand effect.

Such oligonucleotides can be introduced into the cells using the above-mentioned recombinant virus approach, where the second sequence carried by the virus is the oligonucleotide sequence. Another possibility is to use antibodies specific for these proteins to inhibit their intracellular signaling activity. It is possible that these new proteins that have an extracellular domain as well as an intracellular one, the latter can bind to the TNF-R or FAS-R binding domain, and thus antibodies generated to their extracellular domains can be used to block their TNF - or FAS-R ligand-related functions.

Yet another way to inhibit the TNF or FAS-R ligand effect is by the recently developed ribozyme supplementation. Ribozymes are catalytic RNA molecules that specifically cleave RNA. Ribozymes can be designed to cleave the desired target RNA, for example mRNA encoding the new proteins according to the invention or mRNA encoding p55IC, p55DD, FAS-IC or FAS-DD. Such ribozymes will have a sequence specific to the mRNA in question and will be able to interact with it (complementary binding) followed by cleavage of the mRNA to result in a reduction (or complete loss) of the expression of the desired protein to inhibit, where the level of reduced expression is dependent on the level of ribozyme expression in the target cell. To introduce ribozymes into the cells in question (for example those carrying TNF-R or FAS-R), any suitable vector can be used, for example plasmid, animal virus (retrovirus) vectors, which are commonly used for this purpose (see also (i) above, where the virus has as a second sequence a cDNA that codes for the relevant ribozyme sequence). Furthermore, ribozymes can be constructed which have multiple targets (multi-target ribozymes) which can be used, for example, to inhibit the expression of one or more of the proteins according to the invention and/or p55IC, p55DD, FAS-IC or FAS-DD as well ( for reviews, methods etc. regarding ribozymes see Chen et al., 1992; Zhao and Pick, 1993; Shore et al., 1993; Joseph and Burke, 1993; Shimayama et al. 1993; Cantor et al., 1993; Barinaga, 1993;

Crisell et al., 1993 and Koizumi et al., 1993).

(iii) They can be used to isolate, identify and clone other proteins capable of binding them, for example other proteins involved in the intracellular signaling process downstream of the TNF-R or FAS-R intracellular domain. In this situation, these options, namely the DNA sequences encoding them, can be used in the yeast two-hybrid system (see example 1 below) in which the sequence of these proteins will be used as "bait" to isolate, clone and identify from cDNA or genomic DNA libraries other sequences ("swaps") that encode proteins that can bind to these new TNF-R or FAS-R intracellular domain binding proteins. In the same way, it can also be determined whether the specific proteins according to the invention, namely those that bind to p55IC, p75IC or FAS-IC, can bind to other receptors of the TNF/NGF superfamily of receptors. For example, it has recently been reported (Schwalb et al., 1993; Båens et al., 1993; Crowe et al., 1994) that other TNF-Rs exist in addition to p55 and p75 TNF-Rs. Consequently, by using the yeast two-hybrid system, it can be specifically tested whether the proteins according to the present invention are capable of specifically binding to these other TNF-R or other receptors in the TNF/NGF superfamily. Furthermore, this approach can also be used to determine whether the proteins according to the present invention are capable of binding to other known receptors in which activity they may have a functional role. (iv) The new proteins can also be used to isolate, identify and clone other proteins in the same classes, i.e. those that bind to TNF-R or FAS-R intracellular regions or to functionally related receptors, and are involved in the intracellular signaling process. In this application, the yeast two-hybrid system indicated above can be used, or the recently developed (Wilks et al., 1989) system that uses non-stringent southern hybridization followed by PCR cloning can be used. In the Wilks et al. publication, the identification and cloning of two putative protein-tyrosine kinases using non-stringent southern hybridization followed by cloning by PCR based on the known sequence of the kinase motif, an evolved kinase sequence, is described. This approach can be used according to the present invention as the use of the sequences of the new proteins to identify and clone the related to TNF-R, FAS-R or related receptor (TNF/NGF superfamily receptors) intracellular domain binding proteins. (v) Yet another approach to using the new proteins according to the invention is to use them in methods for affinity chromatography to isolate and identify other proteins or factors to which they are capable of binding, for example other receptors related to TNF-R (TNF/NGF receptor superfamily) or other proteins or factors involved in the intracellular signaling process. In this application, the proteins according to the invention can be individually bound to affinity chromatographic matrices and then brought into contact with cell extracts or isolated proteins or factors believed to be involved in the intracellular signaling process. After the affinity chromatography procedure, the proteins or factors that bind to the new proteins according to the invention can be eluted, isolated and characterized. (vi) As noted above, the new proteins according to the invention can also be used as immunogens (antigens) to produce specific antibodies. These antibodies can be used for purification purposes of the new proteins either from cell extracts or from transformed cell lines that produce them. Furthermore, these antibodies can be used for diagnostic purposes to identify disturbances related to abnormal functions of the TNF or FAS-R ligand system, for example overactive or underactive TNF- or FAS-R ligand-induced cellular effects. Thus, such disturbances will be related to malfunctioning intracellular signaling systems involving the new proteins, and the antibodies will thus serve as important diagnostic tools.

It must also be noted that the isolation, identification and characterization of the new proteins according to the invention can be carried out using any known standard screening procedure. For example, one of these screening procedures, the yeast two-hybrid procedure as indicated in the following examples (Examples 1 and 3) was used to identify the new proteins according to the invention. Likewise, as indicated above, other procedures may be used such as affinity chromatography, DNA hybridization procedures, etc., which are well known in the art, to isolate, identify and characterize the new proteins of the invention or to isolate, identify and characterize additional proteins, factors, receptors etc. which are able to bind to the new proteins according to the invention or to the receptors belonging to the TNF/NGF family of receptors.

With regard to the antibodies mentioned here, the term "antibody" throughout is intended to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form , as well as fragments thereof, which are provided by any known technique such as, but not limited to, enzymatic cleavage, peptide synthesis or recombinant techniques.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. A monoclonal antibody contains a substantially homogeneous population of antibodies specific for antigens, whose populations contain substantially corresponding epitope binding sites. MAbs can be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature. 256:495-497 (1975); US Patent No. 4,376,110; Ausubel et al., ed. Harlow and Lane ANTIBODffiS: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., ed. Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Juterscience N.Y., (1992,1993), the contents of which are incorporated by reference in their entirety. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridoma producing a mAb according to the present invention can be cultured in vitro, in situ or in vivo. Production of high titers of mAbs in vivo or in situ makes this the currently preferred method of production.

Chimeric antibodies are molecules in which different parts are derived from different animal species, such as those that have the variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies are primarily used to reduce the immunogenicity in use and to increase the yield during production, for example where murine mAbs have higher yields from hybridomas, but higher immunogenicity in mammals, so that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proe. Ntl. Acad. Sei. USA 81:3273-3277 (1984); Morrison et al., Proe.

Nati. Acad. Pollock. USA 81:6851-6855 (1984); Boulianne et al., Nature 312:643-646

(1984); Cabilly et al., European Patent Application 12023 (published Nov. 14, 1984); Neuberger et al., Nature 314:268-270 (1985): Taniguchi et al., European Patent Application 171496 (published February 19, 1985); Morrison et al., European Patent Application 173494 (published March 5, 1986); Neuberger et al., PCT Application WO 8601533 (published March 13, 1986); Kudo et al., European Patent Application 184187 (published June 11, 1986); Sahagan et al., J. Immunol. 137:1066-1074 (1986); robison et al., International Patent Application No. WO8702671 (published May 7, 1987); Liu et al., Proe. Nati. Acad. Say C/&484:3439-3443 (1987); Sun et al., Proe. Nati. Acad. Sei USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988); and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra. These references are in their entirety. incorporated herein by reference.

An anti-iodiotypic (anti-Id) antibody is an antibody that recognizes unique determinants generally associated with the antigen binding site of an antibody. An Id antibody can be produced by immunizing a mammal of the same species and genetic type (eg mouse strain) as the source of the mAb with the mAb to which an anti-Id has been produced. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (anti-Id antibody). See, for example, US Patent No. 4,699,880, incorporated herein in its entirety by reference.

The anti-Id antibody can also be used as an "immunogen" to induce an immune response in another animal to produce a so-called anti-anti-Id antibody. The anti-anti-Id may be epitopically identical to the original mAb that induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies with identical specificity.

Accordingly, mAbs generated against IC-binding proteins, analogs or derivatives thereof according to the present invention or p55IC, p55DD, FAS-IC, FAS-DD, analogs or derivatives thereof can be used to induce anti-Id antibodies in appropriate animals, such as BALB /c mouse. Spleen cells from such immunized mice are used to produce anti-Id hybridomas that secrete anti-Id mAbs. Furthermore, anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to further immunize BALB/c mice. Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for an epitope of the above-mentioned IC-binding proteins, analogs or derivatives or p55IC, p55DD, FAS-IC or FAS-DD.

Anti-Id mAbs thus have their idiotypic epitopes or "idiotopes" structurally similar to the epitope being evaluated, such as GRB protein-a.

The term "antibody<1> is also intended to include both intact molecules as well as fragments thereof, for example, Fab and F(ab')2, which are capable of binding antigen. The Fab and F(ab')2 fragments lacks the Fc fragment in the intact antibody, is more easily removed from circulation and may have less non-specific tissue binding than an intact antibody (Wahl et al. 1, J. Nucl. Med. 24:316-325 (1983)).

It must be noted that Fab and F(ab')2 and other fragments of the antibodies useful in the present invention can be used for detection and quantification of the IC-binding proteins or p55IC, p55DD, FAS-IC or FAS-DD according to the method described here for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab)2 fragments).

An antibody is said to be "capable of binding" a molecule if it is able to specifically react with the molecule to thereby bind the molecule to the antibody. The term "epitope" is intended to refer to the portion of any molecule capable of being bound by an antibody that can also be recognized by that antibody. Epitopes or

"antigenic determinants" usually consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics.

An "antigen" is a molecule or part of such a molecule capable of being bound by an antibody which is further capable of inducing an animal to produce antibody capable of binding to an epitope of it the antigen. An antigen can have one or more than one epitope. The specific reaction referred to above is intended to indicate that the antigen will react in a highly selective manner, with its corresponding antibody and not with a plurality of other antibodies that may be elicited by other antigens.

The antibodies, including fragments of antibodies useful according to the present invention, can be used to quantitatively or qualitatively detect IC-binding proteins or p55IC, p55DD, FAS-JD, FAS-DD in a sample or to detect the presence of cells expressing IC -binding proteins according to the invention or the p55IC, p55DD, FAS-IC, FAS-DD proteins. This can be achieved by immunofluorescence techniques using a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric or fluorometric detection.

The antibodies (or fragments thereof) which are useful according to the present invention can be used histologically, as an immunofluorescence or immunoelectron microscopy for in situ detection of IC-binding proteins according to the invention or p55IC, p55DD, FAS-IC, FAS-DD. In situ detection can be performed by removing a histological sample from a patient and providing a labeled antibody according to the present invention to obtain such a sample. The antibody (or fragment) is preferably obtained by adding or overlaying the labeled antibody (or fragment) onto a biological sample. By applying such a procedure, it is possible to determine, not only the presence of the IC-binding proteins or p55IC, p55DD, FAS-IC, FAS-DD, but also its distribution in the examined sample. In applying the present invention, one skilled in the art can readily appreciate that any of several histological methods (such as staining procedures) can be modified to achieve such in situ detection.

Such assays for IC-binding proteins according to the present invention or pS5IC, p55DD, FAS-IC, FAS-DD, typically comprise incubation of a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in the tissue culture, in the presence of a detectably labeled antibody capable of identifying the IC-binding proteins or pSSIC, p55DD, FAS-IC, FAS-DD, and detecting the antibody at any one of a number techniques well known in the art.

The biological sample may be treated with a solid phase support or carrier such as mtrocellulose, or other solid support or carrier capable of immobilizing cells, cell particles or soluble proteins. The support or support may then be washed with appropriate buffer followed by treatment with a detectably labeled antibody of the present invention as noted above. The solid phase support or carrier can then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on each solid support or carrier can then be detected by conventional means.

By "solid phase support", "solid phase carrier", "solid support", "solid support", "support" or "carrier" is meant any support or carrier capable of binding antigen or antibodies. Well-known supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite. The nature of the carrier can either be soluble to a certain extent or insoluble for the purposes of the present invention. The support material can have almost any structural configuration as long as the linked molecule is capable of binding to an antigen or antibody. Thus, the support or carrier may be spherical, as in a bead, cylindrical, like the inner surface of a test tube, or the outer surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or supports include polystyrene beads. The person skilled in the art will understand which other suitable carriers are relevant for the binding of antibody or protein, or will be able to provide these for use in ratine experiments.

The binding activity of a given batch of antibody according to the invention as noted above can be determined according to well-known methods. The person skilled in the art will be able to determine operative and optimal analysis conditions for each determination using routine experiments.

Other such steps as washing, stirring, shaking, filtering and the like may be added to the analyzes as usual or necessary for the particular situation.

One of the ways in which the antibodies can be detectably labeled is by binding the same to an enzyme and using an enzyme immunoassay (EIA). This enzyme will in turn, when later exposed to a suitable substrate, react with the substrate in such a way as to produce a chemical group which can be detected, for example by spectrophotometric, fluorometric or visual means. Enzymes that may be useful for detectable labeling of the antibody include, but are not limited to, malate hydrogenase, staphylococcal nuclease, delta 5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, araparinase, glucose oxidase, beta-galactosidase, ribonuclease , urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection can be carried out by colorimetric methods that use a chromogenic substrate for the enzyme. Detection can also be achieved by visual comparison of the degree of enzymatic reaction of a substrate compared to similarly prepared standards.

Detection can be achieved using any of a variety of other immunoassays. For example, by radioactivity labeling the antibodies or antibody fragments, it is possible to detect R-PTPase using a radioimmunoassay (RIA). A good description of RIA can be found in Laboratory, Techniques and Biochemistry in Molecular Biology, by Works, T.S. et al., North Holland Publishing Company, NY (1978) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., which is incorporated herein by reference. The radioactive isotope can be detected by such means as the use of a y counter or a scintillation counter or by autoradiography.

It is also possible to label an antibody according to the present origin with a

fluorescent compound. When the fluorescently labeled antibody is exposed to light of the appropriate wavelength, its presence can be detected due to fluorescence. Among the commonly used fluorescently labeled label compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, pycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence-emitting metals such as ^^ E, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent labeled antibody is then determined by detecting the presence of luminescence that occurs during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeled compounds are luminol, isoluminol, terromatic acridinium ester, imidazole, acridinium salt and oxalate esters.

Likewise, a bioluminescent compound can be used to label the antibody according to the present invention. Bioluminescence is a type of chemiluminescence found in biological systems where a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.

An antibody molecule according to the present invention can be adapted for use in an immunometric analysis, also known as a "two-site" or "sandwich" analysis. In a typical immunometric assay, an amount of unlabeled antibody (or fragment of antibody) is bound to a support or support and an amount of detectably labeled soluble antibody is added to allow detection and/or quantification of the ternary product formed between the solid-phase antibody, the antigen, and the labeled antibody.

Typically, and preferably, immunometric assays comprise "forward" assays where the antibody bound to the solid phase is first brought into contact with the sample being tested to extract the antigen from the sample by forming a binary solid phase antibody-antigen complex. After an appropriate incubation period, the solid support or support is washed to remove the remainder of the fluid sample including unreacted antigen, if any, and then contacted with a solution containing an unknown amount of labeled antibody (acting as a "reporter molecule"). After a second incubation period to allow the labeled antibody to complex with the antigen bound to the solid support or support through the unlabeled antibody, the solid support or support is washed a second time to remove the unreacted labeled antibody.

In another type of "sandwich" analysis which can also be used with the antigens according to the invention, so-called "simultaneous" and "reverse" analyzes are used. A simultaneous assay involves a single incubation step where the antibody bound to the solid support or carrier and the labeled antibody are both added to the sample being tested at the same time. After incubation is complete, the solid support or carrier is washed to remove residual fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with a solid support or carrier is then determined as if it were a conventional "forward" sandwich assay.

In the "reverse" assay, a stepwise addition first of a solution of labeled antibody to the fluid sample is used followed by the addition of unlabeled antibody bound to a solid support or carrier after an appropriate incubation period. After a second incubation, the solid phase is washed in a conventional manner to free it of the remainder of the sample being tested and the solution of unreacted labeled antibody. The determination of the labeled antibody associated with a support or carrier is determined as in the "simultaneous" and the "forward" assay.

The new proteins according to the invention can, once they have been isolated, identified and characterized by standard screening procedures, for example the yeast two-hybrid method, affinity chromatography and other well-known methods, be produced by any standard recombinant DNA procedure (see for example Sambrook et al., 1989) where appropriate eukaryotic or prokaryotic host cells are transformed by appropriate eukaryotic or prokaryotic vectors containing the sequences encoding the proteins. Consequently, the invention also includes such expression vectors and transformed hosts for the production of the proteins according to the invention. As mentioned above, these proteins include their biologically active analogs and derivatives and thus the vectors encoding them also including the vectors encoding the analogs of these proteins and the transformed hosts including those that produce such analogs. The derivatives of these proteins are the derivatives produced by standard modification of the proteins or their analogues, produced by transformed hosts.

According to another aspect, the invention relates to the use of the free intracellular region of p55 TNR-R (p55IC) or FAS-R (FAS-IC) or their so-called "death regions" for the preparation of a drug for the treatment of specific disorders (p55DD or FAS, respectively -DD as a means of increasing the TNF or FAS-R ligand effect on cells, or on its own (see example 2). Where it is desirable to introduce a TNF- or FAS-R ligand-induced cytotoxic effect in cells, for example cancer cells or HIV-infected cell, p55IC, p55DD, FAS-IC or FAS-DD can be introduced into such cells using the above-mentioned (see (i) above) recombinant animal virus (eg vaccinia) approach. Here also the native p55IC , p55DD, FAS-IC or FAS-DD, biologically active analogues and derivatives or fragments thereof, can be used, all of which can be prepared as indicated above.

Likewise, the present invention also enables the specific blocking of the TNF effect or the FAS-R ligand effect by blocking the activity of p55IC, p55DD, FAS-IC or FAS-DD, for example antisense oligonucleotides can be introduced into the cells by blocking the expression of p55IC, p55DD, FAS-IC or FAS-DD.

The present invention also relates to pharmaceutical compositions comprising recombinant animal virus vectors encoding the TNF-R or FAS-R intracellular domain binding proteins (including p55IC, p55DD, FAS-IC and FAS-DD), which vector also encodes a virus surface protein capable of to bind specific target cell (eg cancer cells) surface proteins to direct the insertion of the intracellular site-binding protein sequences into the cells.

According to another aspect, the present invention also specifically relates to the effects of the self-associating intracellular regions of the p55 TNF receptor (p55-IC, see example 2). An example of such effects which is an effect normally mediated by TNF binding to its receptor and which is mimicked by the signaling activity of the self-associating p55-IC or parts thereof, is the induction of expression of the gene that codes for IL-8. IL-8 is a cytokine belonging to the subclass of chemokines that have primary chemotactic activity, and has been shown to play a key role in the chemotaxis of granulocytes and other cell types associated with a number of pathological conditions (see, for example, Endo et al., 1994; Sekido et al., 1993; Harada et al., 1993; Ferrick et al., 1991).

TNF has a beneficial activity and is used as such in treatment to destroy tumor cells and virus-infected cells or to increase the antibacterial activities of granulocytes. However, as indicated above, TNF also has undesirable activities in cases where it is desirable to block its activity, including those situations where large doses of TNF are used in cancer therapy, antiviral therapy or antibacterial therapy.

Accordingly, it is desirable to be able to direct TNF or a substance capable of mimicking its beneficial activity to the cells or tissues specifically desired to be treated.

According to the present invention, it has been found that the self-associating intracellular region of p55-R (p55-IC) can, in a ligand-independent manner, mimic a number of the effects of TNF, for example the "death region" of p55-IC can induce cytotoxic effects on cells and that p55-IC can induce BL-8 gene expression. Thus, it is possible to use p55-IC to mimic TNF function in a site-directed manner, i.e. to introduce pSS-IC only in the cells or tissues it is desired to treat.

Another example of the above-mentioned approach as mentioned above is to specifically transfect (transform) tumor cells or malignant tissue with a DNA molecule encoding pSS-IC or a part thereof which can induce not only cytotoxic effects on such cells or tissue, but also increase these effects by so-called co-induction of IL-8, which will result in the accumulation at the site of these cells or tissues of granulocytes and other lymphocytes, which will in turn serve to destroy the tumor cells or tissues. This approach avoids the need for administration of large doses of TNF with its associated harmful side effects.

Using conventional recombinant DNA technology, it is possible to produce different regions of v p55-IC and to determine which region is responsible for each TNF-induced effect, for example, it has been found that the "death region" is responsible for cytotoxicity (Example 2) , and various other constructs have already been produced containing parts of p55-IC, which parts (together with parts or the entire death region) may be responsible for other TNF effects, and which can be used in a ligand-independent manner once it is self-associated for activity, to induce these effects, for example IL-8 induction.

It must be noted that the sequence of p55-IC involved in the induction of other TNF-associated effects (for example EL-8 induction) may be different from that involved in cytotoxicity, i.e. may include none or only parts of the "death region" and have other sequence motifs from other regions of the intracellular area or may be the same sequence, different properties of the sequence (same sequence motif) being involved in the induction of different effects.

Accordingly, as detailed above and below, expression vectors containing these p55-IC portions, analogs or derivatives thereof can be prepared, expressed in host cells, purified and tested for their activity. In this way, a number of such p55-IC fragments having one or more TNF-associated activities can be prepared and used in a differential manner for the treatment of a large number of pathological conditions, for example viral infections, bacterial infections, tumors, etc. In all in these situations, the specific activity can be increased by incorporation (or co-transfection) with the p55-IC fragment responsible for IL-8 gene expression induction, allowing the desired IL-8 chemotactic activity to increase the destruction of the cells or tissues it is desired to destroy .

Thus, without systemic administration of TNF, it is possible to induce its desired effects by specifically introducing all or parts of p55-IC into the cells or tissues it is desired to treat.

p55-IC can be introduced specifically into the cells or tissues it is desired to destroy by any of the above procedures. For example, one way of doing this is to form a recombinant animal virus, for example derived from Vaccinia, into which DNA the following two genes will be introduced: the gene encoding a ligand that binds to the cell surface proteins specifically expressed by the cells, for example, such as the AJDS virus gp 120 protein that binds specifically to some cells (CD4

lymphocytes and related white blood cells) or optionally other ligands that bind specifically to cells bearing TNF-R, so that the recombinant viral vector may be able to bind to such TNF-R bearing cells; and the gene encoding p55-IC or part thereof. Thus, the expression of cell surface binding protein on the surface of the virus will target the virus specifically to the tumor cell or other TNF-R-bearing cell, after which p55-IC or parts thereof encoding the sequence will be introduced into the cell via

the virus and immediately expressed in the cells, it will result in an increase in the TNF effect which leads to the killing of the tumor cells or other TNF-R bearing cells it is desirable to kill or the induction, for example, of IL-8 which will lead to cell death. Construction of such recombinant animal virus by standard procedures (see, for example, Sambrook et al., 1989). Other possibilities are to introduce the sequences of p55-IC or parts thereof in the form of oligonucleotides that can be absorbed by the cells and expressed therein.

The present invention thus specifically relates to pharmaceutical compositions comprising the above-mentioned recombinant animal virus vectors that encode p55-IC or parts thereof, whose vector also encodes a virus surface protein that is capable of binding specific target cell (for example cancer cells) surface proteins to direct insertion of p55-IC or parts thereof, sequences into the cells.

It can also be pointed to new synthetic TNF receptors that are soluble and capable of oligomerization to form dimers, and possibly also higher order multimers, TNF receptor molecules, where each monomeric part of these receptors is capable of forming a TNF monomer. TNF naturally acts as a homotrimer containing three active TNF monomers, each capable of binding to a single TNF receptor molecule, while the TNF receptors normally act as monomers, each capable of binding only to one of the monomers of the TNF homotrimeric molecule. Thus, when TNF binds to the TNF receptors on the cell surface, it is able to bind to three receptor molecules to result in clustering of the TNF receptors which is believed to be the start of the signaling process that ultimately triggers the observed TNF effect on the cells.

While TNF has many desired effects such as the ability to destroy, for example, tumor cells or virus-infected cells and to increase the antibacterial activities of granulocytes, however, TNF has many undesirable effects such as, for example, in many serious diseases including autoimmune diseases, rheumatoid arthritis , graft-versus-host reaction (implant destruction), septic shock, TNF has been implicated as the main cause of the pathological tissue destruction. TNF can also cause large weight loss (cachexia) by suppressing the activities of adipocytes. However, even when administered for the desired activity, for example for the treatment of various malignant or viral diseases, the dosage of TNF used is often high enough to cause a number of undesirable side effects in the patient, for example destruction of healthy tissue.

Consequently, in all of the above-mentioned cases where TNF action is undesirable, an effective inhibitor of TNF has been sought. Many TNF blocking agents have been proposed, including soluble proteins capable of binding TNF and inhibiting its binding to its receptors and thus inhibiting the cytotoxic effects of TNF (see EP 308378, EP 398327 and EP 568925). However, these TNF binding proteins, or soluble TNF receptors, are monomers that each bind only one TNF monomer of the TNF homotrimer. Thus, the blocking of the TNF function cannot be complete as each monomeric receptor-bound TNF molecule still has two TNF monomers which are freely able to bind cell surface TNF receptors and produce its effects on the cells.

In order to overcome the above-mentioned disadvantages of blocking TNF function, means have been developed for constructing, as fusion proteins, soluble oligomeric TNF receptors which are capable of binding at least two TNF monomers of the naturally occurring TNF homotrimer molecule. As a consequence, these soluble TNF receptors bind more strongly to their TNF ligand than previously known monomer soluble TNF binding proteins or receptors. For example, when the soluble TNF receptor according to the invention is in the form of a dimer, it is able to bind two TNF monomers of a TNF triner and thus cause a more complete neutralization of TNF, where the neutralization is more persistent due to lower dissociation rate of the dimeric soluble receptors from TNF. Furthermore, such soluble, oligomeric receptors are also larger than their monomeric counterparts and thus pharmaceutically also have advantages due to the likelihood that they have a slower rate of excretion from the body.

The basis for the development of the soluble oligomeric TNF receptors was the discovery that the intracellular region of the p55-R TNF receptor was capable of self-association and further that within this intracellular region (p55-IC) there exists a region, the so-called "death region" , which is also capable of self-association and as such in a ligand-independent manner, can cause cytotoxic effects on the cells (see example 2). By using this self-association property of p55-IC and its "dead region" it is thus possible to construct a fusion protein using standard recombinant DNA technology containing essentially the entire extracellular region of a TNF receptor such as p75-R or p55-R receptors, preferably p55-R, and fused thereto, essentially the entire extracellular region (p55-IC) or the death region of p55-IC. In this way, a new fusion product was produced which has at one end the TNF binding region, i.e. the extracellular region of the receptor, and at its other end the intracellular region or dead region thereof which is capable of self-association. Accordingly, such a product can oligomerize by self-association between two (and possibly more) p55-IC or dead regions thereof to give oligomers (or at least dimers) having at least two TNF binding regions.

Furthermore, it has also been discovered that the Fas/APO1 receptor has a self-associating, intracellular region including a self-associating "death region" which has some homology to p55-IC and the death region thereof (Example 2). Accordingly, it is possible to construct the soluble, oligomeric TNF receptors by fusing the extracellular region of the TNF receptor (as indicated above) to the intracellular region or "dead region" of the Fas/APO1 receptor.

In both of the above two situations, the oligomeric TNF receptors are soluble in that they only have the soluble extracellular region of the TNF receptor and the soluble intracellular region or dead region thereof of either the pS5-R TNF receptor or the Fas/APO1 receptor, i.e. they contain not the transmembrane (insoluble) region of any of these receptor types.

The construction of the above-mentioned oligomeric TNF receptors is described in detail below in example 4. However, it must be noted that after construction of the oligomeric TNF receptors, a situation may arise that has not previously been reported, i.e. that the extracellular region of the TNF receptor is capable of self-association, a situation which is not desirable as it may interfere with the ability of the oligomeric receptor to bind to two or more TNF monomers of the TNF homotrimeric molecules or lead to less than optimal binding of such TNF monomers. Consequently, it is possible in such a situation by standard recombinant DNA procedures to modify the extracellular region of the TNF receptor by, for example, deletion or substitution of one or more amino acid residues contained in the self-associating region to prevent such self-association. Such modifications of the extracellular region of the TNF receptor are thus part of the present invention and are indicated here as analogues or derivatives of the extracellular region of the TNF receptor. In a similar way, the self-associating intracellular region (IC) or the death region (DD) thereof of the p55 receptor or the Fas/AP1 receptor used in the oligomeric TNF receptors can also be analogues or derivatives thereof, i.e. can be any which preferably modification of the p55-IC sequence or parts thereof including the death region (p55DD), or any modification of the Fas/APOl intracellular region (FAS-IC) sequence or parts thereof including the death region (FAS DD) provided that these modifications provide a self -associating product.

Correspondingly, immediately produced and purified, the soluble oligomeric TNF receptors, analogues or derivatives thereof, can be further modified by standard chemical means to give salts and functional derivatives thereof for the purpose of preparing pharmaceutical compositions containing as active ingredients these TNF receptors.

For the production of the soluble, oligomeric TNF receptors, the DNA sequences that code for the extracellular region of the TNF receptor are obtained from existing clones of the entire TNF receptor, thus the intracellular region becomes the death region thereof, and is also the intracellular region or the death region of Fas/ the APO1 receptor (see example 2 and example 5). In this way, the DNA sequence of the desired extracellular region is ligated to the DNA sequence of the desired intracellular region or part thereof including the dead region, and this fused protein is inserted (and ligated) into a suitable expression vector under the control of the promoter and other expression-controlling sequences. Once formed, the expression vector is introduced (transformation, transfection, etc.) into a suitable host cell which then expresses the vector to yield the fusion product of the invention which is the soluble self-associating TNF receptor molecules. These are then purified from the host cells by standard procedures to yield the final product which is the soluble, oligomeric TNF receptors.

The preferred preparation of the fusion product that codes for the extracellular region and the intracellular region or parts thereof is by means of PCR technology using oligonucleotides that are specific for the desired sequences to be copied from the clones that code for the entire TNF receptor molecule. Other means are also possible, such as isolating the desired portions encoding the extracellular region and the intracellular region by restriction endonucleases and then splicing these together in a known manner, with or without modifications at the terminal ends of the restriction fragments to ensure correct fusion of the desired parts of the receptor (extracellular and intracellular regions or parts thereof). The thus obtained fusion products are then inserted into the desired expression vector.

In a similar way, soluble, oligomeric Fas/APO1 (FAS) receptors containing the extracellular region of the Fas/APO1 receptor and the self-associating intracellular region of p55-R (p55-IC), the death region thereof (p55DD), or the self-associating intracellular region of the Fas/APO1 receptor

(FAS-IC) or its dead area (FAS DD), or any analogues or derivatives thereof (see above). The construction of these soluble, oligomeric FAS receptors is described in detail in Example 5 below, using an available cloned full-length FAS receptor coding sequence as starting material and the appropriate oligonucleotides for PCR production of the desired extracellular and intracellular regions, followed by ligation thereof to yield a fusion product which can then be inserted into an appropriate expression vector. As indicated above and below, prokaryotic or eukaryotic vectors and host cells can be used to produce the desired oligomeric FAS receptors, which can then be purified and formulated, as an active ingredient, in a pharmaceutical composition.

The above-mentioned soluble, oligomeric FAS receptors are intended for effective blocking of the Fas ligand, which also exists as a trimer (similar to TNF, see above), where each oligomeric receptor is able to bind two or possibly more Fas ligands and thereby neutralize their activity. The Fas ligand is known to be mainly cell surface associated, but can also exist in a soluble form. In any case, the oligomeric FAS receptors can bind at least two monomers of this ligand and thereby neutralize more effectively (than monomeric FAS receptors) the activity of the Fas ligand. The Fas ligand, and thus activation thereby of the FAS receptor, has been seen in the context of a number of pathological conditions, particularly those involving liver damage (apoptosis of hepatocytes, for example), including liver damage associated with hepatitis, as well as in autoimmune conditions, including lymphocyte damage (apoptosis) in HIV-infected humans (see, for example, Ogasawara et al., 1993, Cheng et al., 1994). Accordingly, the soluble, oligomeric FAS receptors are intended for blocking the activity of the Fas ligand and can be used as an active ingredient in pharmaceutical compositions for the treatment of such Fas ligand-related pathological conditions.

Likewise, soluble, oligomeric receptors that have binding affinity for both TNF and FAS-R ligand, the so-called "mixed" TNF-R/FAS-R oligomeric receptors, are also made possible. These mixed oligomeric receptors will contain at least one TNF-R extracellular region and at least one FAS-R extracellular region linked to the oligomeric receptor by each of these extracellular regions fused to any of the above, even -associating p55IC, p55DD, FAS IC or FAS DD.

These mixed oligomeric receptors can be prepared by: (a) providing any of the above fusion products containing the extracellular region of a TNF-R (p75 TNF-R or preferably p55 TNF-R) fused to any of the self-associating intracellular regions p55 IC and FAS IC or any of the self-associating "death" regions p55DD and FAS DD, or self-associating parts, analogs or derivatives thereof; (b) providing any of the above fusion products containing the extracellular region of FAS-R fused to any of the self-associating P55IC, FAS-IC, p55DD and FAS DD, or any self-associating portions; analogues or derivatives of any of these; and (c) mixing any of the TNF-specific fusion products of (a) with any of the FAS-R ligand-specific fusion products of (b) to provide (after standard selection and purification procedures) oligomers (dimers or higher order oligomers) receptors having at least both extracellular regions of TNF-R and FAS-R linked by the self-association of their fused IC or DD regions.

A second possibility for producing the above-mentioned mixed oligomeric receptors is by co-transformation of suitable host cells with the above-mentioned expression vectors, one of which codes for TNF-specific TNF-R fusion products and one of which codes for FAS-R ligand-specific FAS- R fusion products. After the expression of these different fusion products in the host cells, the mixed oligomeric (TNF-R/FAS-R) receptors can be obtained by means of standard purification and selection procedures.

The utility of these mixed affinity oligomeric receptors is primarily for neutralization of both the TNF and FAS-R ligands when the endogenous expression is excessive or is at undesirably high levels following exogenous administration. Recent evidence points to the possibility that a synergism exists in function between the FAS-R ligand (usually cell surface-associated) and TNF-α (which may also be cell surface-associated). Accordingly, in some cases it is desirable to neutralize both of these ligands at the same point on the cell surface, i.e. such a mixed affinity receptor can block both TNF binding to its receptor and FAS-R ligand binding to its receptor. Accordingly, these mixed affinity receptors can be used as an active ingredient in pharmaceutical compositions for the treatment of such conditions (see above) where both the TNF and FAS-R ligand effects are undesirable.

Similarly along the lines mentioned above regarding the soluble, oligomeric TNF-R and FAS-R and mixed TNF-R/FAS-R oligomers according to the invention, it is possible to produce soluble, oligomeric receptors for other receptors or any mixtures thereof, in particular those of any other families of the TNF/NGF superfamily. In this case, any of the extracellular regions of the different receptors can be fused to the above-mentioned self-associated intracellular regions or parts thereof or any of the intracellular regions of the superfamily members capable of self-association .

Expression of any of the recombinant proteins according to the invention as mentioned herein can be effected in eukaryotic cells (for example yeast, insects or mammalian cells) using suitable expression vectors. Any method known in the art may be used.

For example, DNA molecules encoding the proteins obtained by any of the methods are inserted into appropriately engineered expression vectors by techniques well known in the art (see Sambrook et al., 1989). Double-stranded cDNA is joined to plasmid vectors by homopolymeric tails or by restriction ligation involving the use of synthetic DNA linkers or blunt ligation techniques. DNA ligases are used to ligate the DNA molecules and undesired binding is entered into by treatment with alkaline phosphatase.

To be able to express the desired protein, an expression vector must also include specific nucleotide sequences containing the transcription and translation regulatory information bound to DNA that codes for the desired protein in such a way as to allow gene expression and production of the protein. First, for the gene to be transcribed, it must have a promoter in front of it that can be recognized by RNA polymerase to which the polymerase binds and thus initiates the transcription process. There are different such promoters in use, which work with different efficiencies (strong and weak promoters). They are different for prokaryotic and eukaryotic cells.

The promoters that can be used in the present invention can be either constitutive, for example the int promoter from bacteriophage A, for example the promoter from the 3-lactamase gene from pBR322 and the CAT promoter from

the chloramphenicol acetyltransferase gene from pPR325 etc. or inducible such as the prokaryotic promoters including the major right and left promoters from bacteriophage A

(Pl and Pr), trp., recA. lacZ. lad, ompF and gal promoters of E. coli, or the trp-lac hybrid promoter, etc. (Glick, B.R., (1987). In addition to the use of strong promoters to generate large amounts of mRNA to achieve high levels of gene expression in prokaryotic cells , it is necessary to also use ribosomal binding sites to ensure that the mRNA is efficiently translated. An example is the Shine-Dalgarno sequence (SD sequence) which is appropriately placed from the initiation codon and complemented to the 3'-terminal sequence of the 16S RNA.

For eukaryotic hosts, different transcriptional and translational regulatory sequences may be used, depending on the nature of the host. They may be derived from viral sources, such as adenovirus, bovine pailloma virus, Simian virus or the like, where the regulatory signals are associated with a particular gene that has a high expression level. Examples are the TK promoter for Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter etc. The transcription initiation regulatory signals can be selected which allow repression and activation so that the expression of the genes can be modulated.

The DNA molecule comprising the nucleotide sequence encoding the fusion product proteins is inserted into a vector having the operably linked transcriptions and translational regulatory signals capable of integration of the desired gene sequences into the host cell. The cells that have been stably transformed by the introduced DNA can be selected by also including one or more markers that allow for selection of the host cells containing the expression vector. The marker can provide phototropy to an auxotropic host, biocide resistance, for example antibiotics, or heavy metals, such as copper or the like. The selectable marker gene can either be linked directly to the DNA gene sequence to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be required for optimal synthesis of termination signals, cDNA expression vectors comprising such elements include those described by Okayama, H. (1983).

In a preferred embodiment, the introduced DNA molecule will be incorporated into a plasmid or viral vector which is capable of autonomous replication in the opposing host. Factors important in choosing a particular plasmid or viral vector include: the ease with which the recipient cells containing the vector can be recognized and selected from the counterproductive cells that do not contain the vector; the number of copies of the vector desired in a particular host; and whether it is desirable to be able to "move" the vector between host cells of different species.

Preferred prokaryotic vectors include plasmids such as are capable of replication in E. coli, for example pBR322, ColE1, pSOO1, ACYC 184 etc. (see Maniatis et al., 1982; Sambrook et al., 1989); Bacillus plasmids such as pC194, pC221, pT127 etc.

(Gryczan, T., (1982)); Streptomyces plasmids including pLT101 (Kendall, J.K. et al.,

(1987)); Streptomyces bakeriophages such as ØC31 (Chater, K.F. et al, in: Sixth International Symposium on Actionomvcetals Biology (1986)) and Pseudomonas plasmids (John, J.F. et al., (1986) and Izaki. K. (1978)). Preferred eukaryotic plasmids include BPV, vaccima SV40.2 micron circle, etc. or their derivatives. Such plasmids are well known in the art (Botstein, D. et al., (1982); Broach, J.R. in: The Molecular Biology of the Yeast Saccharomvces: Life Cycle and Inheritance (1981); Broach, J.R.,

(1982); Bollon, D.P. et al., (1980); Manaiatis, T., in Cell Biolev: A Comprehensive Treatise. Vol. 3: Gene Expression. (1980); and Sambrook et al., 1989).

Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the DNA construct(s) may be introduced into a suitable host cell by any of a variety of suitable means: transformation, transfection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection etc.

Host cells to be used according to the invention can be either prokaryotic or eukaryotic. Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia etc. The most preferred prokaryotic host is E. coli. Bacterial hosts of particular interest include E. coli Kl 2 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli W3110 (F", lambda-, prototropic (ATCC 27325), and other enterobacteria such as Salmonella typhimurium or Serratia marcescens and various Pseudomonas species. Under such conditions the protein will not be glycosylated. The prokaryotic host must be compatible with the replicon and the control sequences in the expression plasmid.

Preferred eukaryotic hosts are mammalian cells, such as human, monkey, mouse and Chinese hamster ovary (CHO) cells as they provide post-translational modifications to protein molecules including proper folding or glycosylation at correct sites. Also, yeast cells can perform post-translational peptide modifications including glycosylation. A number of recombinant DNA strategies exist that use strong promoter sequences and a high number of copies of plasmid can be used for the production of the desired proteins in yeast. Yeast recognizes the leader sequences on cloned mammalian gene products and secretes peptides carrying leader sequences (ie pre-peptides).

After introduction of the vector, the host cells are cultured in a selective medium that selects for growth on vector-containing cells. Expression of cloned gene sequences results in production of the desired proteins.

Purification of recombinant proteins is carried out by any of the methods known for this purpose, i.e. any conventional procedure comprising extraction, precipitation, chromatography, electrophoresis or the like. A further purification procedure which can be used in preference for purification of the protein according to the invention is affinity chromatography using anti-TNF receptor monoclonal antibodies which are produced and immobilized on a gel matrix contained in a column. Impure preparations containing the recombinant protein are passed over the column. The protein will be bound to the column by the specific antibody while the impurities will pass through. After washing, the protein is eluted from the gel by a change in pH or ionic strength.

As used herein (see above), the term "salts" refers to both salts of carboxyl groups and to acid addition salts" of amino groups of the protein molecule formed by means known in the art. Salts of a carboxyl group include inorganic salts, for example, sodium, calcium and salts with organic bases such as, for example, those formed with amines, such as triethanolamine, arginine or lysine Acid addition salts include, for example, salts with mineral acids and salts with organic acids.

"Functional derivatives" as used herein cover derivatives which may be prepared from the functional groups acting as side chains on the residues or the N- or C-terminal groups by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable acceptable, i.e. they do not destroy the activity of the protein and do not give toxic properties to compositions containing them. These derivatives include aliphatic esters or amides of the carboxyl groups and N-acyl derivatives of free amino groups of O-acyl derivatives of free hydroxyl groups formed with acyl groups (for example alkanoyl or carboxylic acid aroyl groups). "Fractions" as used herein refers to any part or portion of the receptor, (intracellular or extracellular regions thereof), or of the proteins that bind to the intracellular region of the receptor, provided that it maintains its biological activity.

As mentioned above, the present invention also relates to various pharmaceutical compositions comprising a pharmaceutically acceptable carrier and various noted active ingredients according to the invention or their salts, functional derivatives or mixtures of any of the foregoing. These compositions may be used in any of the conditions noted herein, for example in conditions where there is an overproduction of endogenous TNF, such as in cases of septic shock, cachexia, graft-versus-host reactions, autoimmune diseases such as rheumatoid arthritis, etc. The mode of administration can be via any accepted route of administration for similar agents and will depend on the condition to be treated, for example when used to inhibit TNF effects they are administered intravenously in the case of septic shock or local injection in the case of rheumatoid arthritis (for for example in the knee), or continuously by infusion, etc. The compositions can also be used, for example, in cases of TNF intoxication caused by exogenous administration of excessive amounts (overdoses) of TNF, for example in the case of cancer therapy or therapy of viral diseases.

The pharmaceutical compositions according to the invention are prepared for administration by mixing the protein or its derivatives with physiologically acceptable carriers, stabilizers and excipients, and prepared in dosage form, for example by lyophilization in a dosage glass. The amount of active compound to be administered will depend on the route of administration, the disease to be treated and the condition of the patient. For example, local injection in inflammatory conditions of rheumatoid arthritis will require less active ingredient on a body weight basis than intravenous infusion in septic shock.

Other aspects of the invention will become apparent from the following examples.

The invention will now be described in greater detail in the following non-limiting examples and the attached figures:

EXAMPLE 1

Cloning and isolation of proteins that bind to the intracellular regions of the p55 and p75 TNF receptors

To isolate proteins that interact with the intracellular regions of the p55 and p75 TNF receptors (p55IC and p75 IC), the yeast two-hybrid system was used (Fields and Song, 1989). Briefly, this two-hybrid system is a yeast-based genetic analysis system for detecting specific protein-protein interactions in vivo by reintroduction of a eukaryotic transcriptional activator such as GAL4 that has two separate regions, a DNA binding and an activation region, which regions when expressed and bound together to form a reintroduced GAL4 protein, is able to bind to an upstream activation sequence which in turn activates a promoter controlling the expression of a reporter gene such as lacZ or HJ.S3, the expression of which is easily observed in the cultured cells. In this system, the genes for the respective interacting proteins are cloned into separate expression vectors. In one expression vector, the sequence of a protein of interest is cloned in phase with the sequence of the GAL4 DNA binding region to generate a hybrid protein with the GAL4 DNA protein region, and in the second vector, the sequence of the second protein of interest is cloned in phase with the sequence of the GAL4 activation region to generate a hybrid protein with the GAL4 activation region. The two hybrid vectors are then co-transformed into a yeast host strain that has a lacZ or HIS3 reporter gene under the control of upstream GAL4 binding sites. Only the transformed host cells (cotransformants) in which the two hybrid proteins are expressed and are able to interact with each other, will be able to express the reporter gene. For the lacZ reporter gene, host cells expressing this gene will turn blue in color when X-gal is added to the cultures. Thus, blue colonies are indicative of the fact that the two cloned candidate proteins are able to interact with each other.

Using this two-hybrid system, the p55IC and p75IC intracellular regions are cloned separately into the vector pGBT9 (carrying the GAL4 DNA-binding sequence, supplied by CLONTECH, USA, see below), to form fusion proteins with the GAL4 DNA-binding region ( correspondingly, the intracellular region, FAS-IC and part of 55IC, namely 55DD, were also cloned into pGBT9 and used to isolate other IC-binding proteins, see example 3 below). For cloning of p55IC and p75IC into pGBT9, clones encoding full-length cDNA sequences of p55 TNF-R (Schall et al., 1990) and p75 TNF-R (Smith et al., 1990) were used from which the intracellular regions (IC) were excised as follows: p55IC was excised using the enzymes EcoRI and SalI, the EcoRI-SalI fragment containing the p55IC sequence was then isolated by standard procedures and inserted into the pGBT9 vector opened at its multiple cloning site (MCS) region, by with the help of EcoRI and Sali. p75 IC was excised using the enzymes BspHI and SalI, the BspHI-Sall fragment containing the p75 IC sequence was isolated by standard procedures and silenced using the Klenow enzyme to generate a fragment that can be inserted into the pGBT9 vector opened with Smal and Sally.

The above hybrid (chimeric) vectors were then countertransfected (separately, one cotransfection with the p55IC hybrid and one with the p75 IC hybrid vector) together with a cDNA library from human HeLa cells cloned with the pGAD GH vector, which carries the GAM activation region, into the HF7c yeast host strain ( all the above-mentioned vectors pGBT9 and pGAD GH carrying the HeLa cell cDNA library and the yeast strain were obtained from Clontech Laboratories, Inc., USA, as part of the MATCHMAKER Two-Hybrid System, #PT1265-I). The co-transfected yeasts were selected for their ability to grow in medium without histidine (His medium), growing colonies indicative of positive transformants. The selected yeast clones were then tested for their ability to express the lacZ gene, i.e. for their LAC Z activity, by adding X-gal to the pellet medium which is catabolized to form a blue colored product by B-gal oxidase, the enzyme encoded by the lacZ gene. Thus, blue colonies are indicative of an active lacZ gene. For activity of the lacZ gene, it is necessary that the GAL4 transcriptional activator is present in an active form in the transformed clones, namely that the GAL4 DNA-binding region encoding one of the above-mentioned hybrid vectors is combined correctly with the GAL4 activator region encoded by the second the hybrid vector. Such a combination is only possible if the two proteins fused to each of the GAL4 regions are capable of stably interacting (binding) with each other. Thus, His<+> and blue (LAC Z<+>) colonies that were isolated were colonies that have been cotransfected with a vector encoding p55IC and a vector encoding a protein product of human HeLa cell origin capable of binding stable to p55IC; or which has been transfected with a vector encoding p75IC and a vector encoding a protein product of human HeLa cell origin capable of binding stably to p75 IC.

Plasmid DNA from the above-mentioned His<+>, LAC Z"<5>" yeast colonies was isolated and electroporated into E. coli strain HB 101 by standard procedures by selecting from Leu<+> and ampicillin resistant transformants, these transformants are the carrying the hybrid GAD GH vector having both the Amp<R> and Leu^ coding sequences. Such transformants are therefore clones carrying the sequences encoding newly identified proteins capable of binding to p55IC or p75IC. Plasmid DNA was then isolated from these transformed E. coli and retested by: (a) retransforming them with the original intracellular site hybrid plasmids (hybrid pGTB9 carrying either the p55IC or p75IC sequences) into yeast strain HF7 as indicated above. As controls, vectors carrying irrelevant protein coding sequences, for example pACT-lamin or pGT9 alone, were used for cotransfection with the p55IC-binding protein or p75IC-binding protein coding plasmids. The cotransformed yeasts were then tested for growth on His medium alone, or with different levels of 3-aminotriazole; and (b) retransformation of plasmid DNA and originally intracellular region hybrid plasmids and control plasmids described in (a) into yeast cells of strain SFY526 and determination of the LAC Z<+> activity (efficiency on B-gal formation, i.e. blue color formation).

The results of the above tests revealed that the growth pattern of the colonies in the His medium was identical to the pattern of LAC Z activity; as measured by the staining of the colony, i.e. the His<+> colonies were also LAC Z<+>. Moreover, the LAC Z activity in liquid culture (preferred culture conditions) was analyzed after transfection with GAL4 DNA binding and activation region hybrids into SFY526 yeast hosts which had a better LAC Z inducibility with the GAL4 transcriptional activator than that of HF-7 yeast host cells.

The results of the above co-transfections are set forth in Table 1 below, from which it is clear that a number of proteins were found capable of binding to pSSIC or p75IC, namely the proteins designated 55.11, which bind to p55IC; and 75.3 and 75.16 that bind to p75IC. All of these p55IC- and p75IC-binding proteins are authentic human proteins that are all encoded by cDNA sequences originating from the HeLa cell cDNA library which was fused to the GAL4 activation region sequence in the plasmid pGAD GH in the above-mentioned yeast two-hybrid analysis system.

Interestingly, it has also been found that fragments of p55IC itself, namely the proteins designated 55.1 and 55.3 were capable of binding to p55IC. These are also discussed in example 2 below.

DNA binding site hybrids:

pGBT9-IC55: full-length intracellular region of p55-TNF-F (p55IC) pACT-lamin: irrelevant protein lamin

pGBT9: vector alone

pGBT9-IC75: full-length intracellular region of p75-TNF-R (p75IC)

Activation range:

55.1 and 55.3 correspond to fragments of the intracellular region of p55-TNF-R 55.11: is the new protein associated with p55-TNF-R

75.3 and 75.16 are the novel proteins associated with p75-TNF-R

The above cloned cDNAs encoding the novel p55IC and p75IC binding proteins, 55.11, 75.3 and 75.16, were then sequenced using standard DNA sequencing procedures. The partial sequences of all these protein coding sequences indicated in fig. 1 a-c, where fig. 1(a) shows the sequence of cDNA encoding protein 55.11; fig. 1(b) indicates the partial sequence of the cDNA encoding protein 75.3; and fig. 1(c) indicates the partial sequence of cDNA encoding protein 75.16.1 fig. 1(d) shows the deduced amino acid sequence of protein 55.11, as derived from the nucleotide sequence in fig. let).

However, it should be noted that a partial sequence of the cDNA encoding the 55.11 protein has also been reported by Khan et al. (1992) in a study of human brain cDNA sequences, whose studies were aimed at the establishment of a rapid and accurate method for sequencing and physical and genetic mapping of human brain cDNA. However, Khan et al. no information regarding the function or other characteristics of the protein encoded by the 55.11 cDNA sequence, such functional or other analyzes were not the purpose of Khan et al. in their study.

Analysis and characterization of the 55. 11 protein

(a) General procedures and materials

(i) Cloning of cDNA of 55. 11

After analysis (eg Northern analysis, see below) of cDNA protein 55.11, it was revealed that the above-mentioned 55.11 cDNA cloned by the two-hybrid screen procedure represented only a partial cDNA 5.11 with nucleotides 925-2863 (see Fig. 1(a) ) which codes for amino acids 309-900 (see Fig. 1(d)). The remaining portion of the 55.11 cDNA [nucleotides 1-924 (Fig. 1(a)) encoding amino acids 1-308 (Fig. 1(d))] was obtained by standard procedures, namely by PCR cloning from a human fetal liver cDNA library (for more details, see below). The full nucleotide sequence of 55.11 (Fig. 1(a)) was determined in both directions by the dideoxy chain termination method.

(ii) Two-hybrid fi-galactosidase expression tests

13-galactosidase expression assays were performed as described above except that in some of the assays the pVP16 vector, which contains the activation region of VP 16, was used instead of pGAD-GH, the Gal4 activation region vector. The numbering of the residues in the proteins encoded by the cDNA inserts is as in the Swiss-Prot databank.

Deletion mutants were produced by PCR, and point mutations by oligonucleotide-directed mutagenesis (Kunkel, 1994).

(iii) Northern analvse

Total RNA was isolated using TRI REAGENT (Molecular Research Center, Inc., Cincinnati, Oh., USA) denatured in formaldehyde/formamide buffer, electrophoresed through an agarose/formaldehyde gel and blotted to a GeneScreen Plus membrane (Dupont, Wilmington, De. , USA) in lOxSSPE buffer using standard techniques. The hybridization blots were hybridized with the partial cDNA of 5.11 (see above, nucleotides 925-2863), radiolabeled with the random-prime kit (Boehringer Mannheim Biochemia, Mannheim, Germany) and washed thoroughly. Autoradiography was performed for 1 week.

(iv) Expression of 55.11 cDNA in HeLa cells and binding of the 55.11 protein to glutathione S-transferase fusion proteins of p55-IC

Glutathione S-transferase (GST) fused to p55-IC (GST-p55IC) and with p55-IC truncated below amino acid 345 (GST-p55IC345) were produced and absorbed onto glutathione-agarose beads as described in Example 2 below (see also Smith and Corcaran, 1994; Frangioni and Neel, 1993). cDNA of 55.11 (1-2863 nucleotides, i.e. full-length 55.11 cDNA) of FLAG-55.11 and of luciferase was expressed in HeLa cells. FLAG-55.11 is the region running between residues 309 and 900 of the 55.11 protein (the partial cDNA of 55.11 (nucleotides 925-2863), originally cloned by two hybrid screen), N-linked to the FLAG octapeptide (Eastman Kodak. New Haven, Ct., USA). Expression of the fusion proteins was done using tetracycline-controlled expression vector (HtTA-1) in a HeLa cell clone expressing a tetracycline-controlled transactivator (see Example 2 below, and Gossen and Bujard, 1992). Metabolic labeling of the expressed proteins with [^S]Met and [^S]Cys (Dupont, Wilmington, De., USA and Amersham, Buckinghamshire, England), lysis of HeLa cells, immunoprecipitation and binding of the labeled proteins with the GST fusion proteins , was performed as described below (Example 2), except that 0.5% instead of 0.1% Nonidet P-40 was present in the cell lysis buffer. The immunoprecipitations of 55.11 and FLAG-55.11 were obtained using rabbit antiserum (diluted 1:500) against a GS fusion protein containing the region of 55.11 extending from amino acids 309 and 900 and a mouse monoclonal antibody against the FLAG octapeptide (M2; Eastman Kodak, 5 µg /ml of cell lysate).

(b) Binding of the 55.11 protein to p55-IC in transformed yeast

In this study, we sought to determine the nature of the binding between 55.11 and p55IC, particularly the regions of both of these proteins involved in this binding. For the above purpose, the two-hybrid procedure was followed where various full-length and deletion mutants of p55IC (see also Example 2 below) in "DNA binding rearranged" constructs were used as "root" to bind to the "prey", which was the partial The 55.11 protein was encoded in constructs in which the partial 55.11 sequence (residues 309-900, as originally isolated) was fused to the "activation region" of the GAL4AD and VP 16AD vectors. Furthermore, various deletion mutants of 55.11 were also constructed and fused to the "activation region" of the GAL4AD vector (for example, mutants of 55.11 with only residues 309-680 and 457-900). The binding of the different "binding region" constructs to the different "activation region" constructs was examined in transfected SFY526 yeast cells. Binding was measured by a two-hybrid 8-galactosidase expression filter assay. The non-relevant proteins SNF1 and SNF4 served as positive controls for the "binding region" and "activation region" constructs; the empty Gal4 (pGAD-GH) and VP 16 (pVP16) vectors served as negative controls for the "activation region" constructs; and the empty Gal4 (pGBT9) vector served as a negative control for the "binding region" constructs. The results of the analysis are set out in Table 2 below where the symbols and "-5-+" indicate the development of strong color within 20-60 minutes before the start of the analysis (positive binding results); and "-" indicates no development of color within 24 hours from the start of the analysis (negative results). Blank fields in the table indicate that binding analysis was not tested. From the results shown in the table above, it can be concluded that 55.11 binds to p55-IC at a site different from the "dead zone" (residues 328-426) of p55-IC.

The 55.11 protein bound to a truncated p55-IC from which the dead region has been deleted (construct 206-328 in Table 2) is more effective than non-truncated p55-IC. It also binds to a further C terminally truncated construct (construct 206-308) and to a construct from which both the dead region and a membrane-proximal part were deleted (construct 243-328). However, 55.11 did not bind the protein to a construct that was N-terminally truncated down to amino acid 266 (Table 2). These findings indicate that the binding site for 55.11 is located in the region between residues 243 and 308 of p55-IC and that the N-terminus of this binding site is between residues 243 and 266.

Transfer of cDNA for 55.11 from the originally cloned "swap" construct containing the Gal4 activation region to a swap construct containing the VP 16 activation region did not reduce the binding efficiency of the 55.11 protein to p55-IC (Table 2). Thus, the structure(s) involved in this binding appeared to be within the 55.11 molecule and did not involve the site of fusion of 55.11 with the activation site.

However, the binding of 55.11 to p55-IC was abolished by even limited truncations of the 55.11 protein at either its C (55.11 construct 309-680) or N terminus (55.11 construct 457-900), (residue 309 is the first residue in the 55.11 protein that is encoded by the partial cDNA clone originally isolated in the two-hybrid study).

The observed binding between 55.11 and p55-IC appeared to be specific as 55.11 did not bind to other proteins, including three receptors of the TNF/NGF receptor family (p75-R, Fas/APO1 and CD40) and other proteins such as lamin and cyclin D ( data not shown). It must be noted that of the other TNF/NGF receptor proteins tested, parts of them comprising their intracellular regions were also tested: human FAS-R (residues 175-319), CD40 (residues 216-277) and p75-TNF-R ( residues 287-461), none of which bound 55.11 (data not shown).

(c) Northern analysis of RNA from several cell lines using 55.11 cDNA as a probe and cloning of full-length 55.11 cDNA

The cell lines examined were HeLa CEM, Jurkat, and HepG2 cells derived from human epithelial carcinoma, an acute lymphoblastic T cell leukemia, an acute T cell leukemia, and a hepatocellular carcinoma. 55.11 cDNA originally isolated (nucleotides 925-2863) was used as a probe. Samples consisted of 10 µg RNA/lane. The results of the Northern analysis are shown in fig. 2 which is a reproduction of a Northern blot.

From fig. 2, it is thus clear that the Northem analysis using 55.11 cDNA as a probe revealed in several cell lines a single hybridization transcript of about 3 kB, which is larger than the cDNA (2 kB) of the originally isolated 55.11 cDNA. Using oligonucleotide primers corresponding to the 55.11 sequence, a 5' extending sequence whose length was around 1 kB was cloned by PCR. The sum of the length of this 5' extended end with that of the originally cloned cDNA was about the length of the 55.11 transcript. The 3 kB cDNA comprising both of these parts was efficiently expressed in transfected HeLa cells (see below) to give a protein of about 84 kDa, suggesting that the 3 kB cDNA contains a translational start site.

(d) In vitro binding of the 55.11 protein to GST fusion proteins containing parts of p55-IC

To ensure that 55.11 can indeed bind to p55-IC and exclude the involvement of yeast proteins in this binding, in vitro interaction of the GST p55-IC fusion proteins produced by bacteria with the protein encoded by the 3 kB 55.11 cDNA (55.11-full) produced by transfected HeLa cells, investigated. In this study, cDNA for full-length 55.11, FLAG-55.11 (residues 309-900 of 55.11 encoded by the original cloned partial cDNA and fused at the N terminus with the FLAG octapeptide), and luciferase (control) were expressed in transfected HeLa cells and metabolically labeled with [3<5>s] Met and [<35>S] Cys. The following proteins were fused to GST: full-length p55-IC (GST-p55-IC) and p55-IC C-terminally truncated up to amino acid 345 (GST-p55-IC345) to remove most of the "dead region" ( see table 2). GST alone served as a control. Lysates of the transfected cells were immunoprecipitated with antibodies against the 55.11 protein when the full-length 55.11 protein was used for binding GST-msion proteins, or with antibodies against the FLAG octapeptide when the FLAG-55.11 fusion product was used for binding the GST fusion proteins. The proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE: 10% acrylamide) followed by autoradiography.

In fig. 3A and B, reproductions of the autoradiograms of the above-mentioned SDS-PAGE gels are shown, where fig. 3A shows the binding of the full-length 55.11 protein (55.11-full) to the various GST fusion proteins; and where fig. 3B shows the binding of the Flag-55.11 fusion product to the various GST fusion proteins. In fig. 3A, a control immunoprecipitate of lysates from cells transfected with germ full-length 55.11 and immunoprecipitated with anti-55.11 antibodies (a55.11 Abs.) is shown in the rightmost lane. In fig. 3B, a control immunoprecipitate of lysates of cells transfected with only FLAG-55.11 and immunoprecipitated with anti-FLAG antibodies (ccFLAG Abs.) is shown in the rightmost lane.

Thus, it is clear from fig. 3A and B that the protein encoded by the full-length 55.11 cDNA can be expressed in HeLa cells and they bind to fusion proteins containing the full p55-IC (GST-p55IC) or a truncated p55-IC lacking most of the death domain (GST- p55IC345) (Fig. 3A). The full-length 55.11 protein did not bind to GST alone (control). Similarly, the HeLa cell-expressed protein encoded by the initially cloned partial cDNA of 55.11 in fusion with FLAG octapeptide (FLAG-55.11) bound in vitro to GST-p55IC and GST-p55IC345, but not to GST (Fig. 3B). The above results therefore also provide further evidence (see (b) above) that 55.11 binds to a region of p55IC upstream of the "dead region", i.e. the region of p55-IC more proximal to the transmembrane region.

Moreover, the above studies demonstrated that antibodies to 55.11 had been successfully produced (Fig. 3A).

(e) Comparison of the deduced amino acid sequence of human 55.11 to those of related proteins present in lower organisms, and the sequence<g>properties of the 55.11 protein

As mentioned above, according to the present invention, the full-length 55.11 cDNA has been cloned and sequenced (see the nucleotide sequence in Fig. 1(a)) and the full-length amino acid sequence of 55.11 has been derived from the cDNA sequence (see the amino acid sequence in Fig. 1( d)). Databank (Genbank™/EMBL Databank) search revealed that parts of the sequence of human 55.11 cDNA (accession numbers T03659, Z19559 and F09128) and its mouse homologue (accession numbers X80422 and Z31147) have already been determined during random sequencing of cDNA libraries. A cDNA sequence (accession number Ul 8247) encoding a human protein of 596 amino acids present in cultures of human hepatoma HC10 cells is equivalent to that of 55.11. However, this hepatoma protein lacks an N terminal portion (amino acids 1-297) corresponding to that of 55.11 and also differs from 55.11 at the regions corresponding to residues 297-377 and residues 648-668 of 55.11. The searches in the data bank also revealed that proteins with very high sequence homology with 55.11 exist in Saccharomyces cerevisiae (yeast), Arabidopsis thaliana (plants) and Caenorhabditis elegans (worms). Thus, 55.11 seems to fulfill an evolutionarily conserved function. In yeast, there are two known proteins (the open reading frame YHR027c and SEN3) whose DNA sequences correspond to that of 55.11. The sizes of both are more that of 55.11. YHR027c is known only by sequencing a genomic clone, while SEN3 has been cloned as a cDNA. The seats within 55.11 that are equivalent to those in SEN3 correlate to the seats for its similarity to YHR027c, although much more similarity is evident between 55.11 and YHR027c than between 55.11 and SEN3. DNA sequence information available for the Arabidopsis thaliana and Caenorhabditis elegans proteins clearly shows, although only partially, that these proteins are equivalent to 55.11 as well as the YHR027c protein from yeast. The only one of these four proteins whose nature has been deduced to date is yeast SEN3 whose homology to 55.11 is limited. SEN3 has been identified as the yeast equivalent of the pl 12 subunit of an activator of the 20S proteasome (the proteolytic core of the 26S proteasome [Rechsteine et al., 1993, DeMartino et al., 1994] (M.R. Culbertson and M. Hockstrasser, personal communication).

In fig. 4 schematically shows a comparison of the deduced amino acid sequence of human 55.11 with that of the above-mentioned related proteins present in lower organisms. In fig. 4, the sequences are compared with the sequences of amino acids derived for: 55.11 cDNA (see Fig. 1(d)), an open reading frame (YHR027c) within a cosmid derived from the 8th chromosome of Saccharomyces cerevisiae (nucleotides 21253-24234, accession number Ul0399) ; SEN3, cDNA of a Saccharomyces cerevisiae protein (accession number L06321); a partial cDNA of a protein from the plant Arabidopsis thaliana (accession number T21500), and a partial cDNA of a protein from the nematode Caenorhabditis elegans (accession number D27396), the "KEKE" sequence in 55.11 is marked with a solid line and the sequence AYAGS(x)gLL with broken lines. The sequences were aligned using the PJJJEUP and PRETTYBOX programs of the GCG package. Gaps introduced to maximize the devices are indicated by dots.

As regards the different sequence characteristics or motifs present in the human

55.11 sequence, the following has been observed: Conserved amino acid sequence motifs were not seen in the protein encoded by 55.11, except for a repetitive "KEKE" sequence found between Lys 614 and Glu 632 (underlined in Fig. 4). Such "KEKE" sequences, which are present in many proteins, include protease subunits and chaperonins, can

promote association of protein complexes (Realini et al., 1994). A sequence AYAGS(x)gLL appears twice in the 55.11 protein (at sites 479, 590, see Fig. 4); no functional significance for this sequence has yet been described.

(f) sequence characteristics of the p55IC region involved in protein binding of 55. 11

As described above (see (b) and (d)), the 55.11 protein binds to a region of p55-IC between residues 243 and 308 (the N terminus of this binding site is between residues 243 and 266), this region being upstream of the "dead region" and more proximally for the transmembrane region of p55-TNF-R. This region within p55-IC to which 55.11 binds has a high content of proline, serine and threonine residues. However, this region does not contain RPM1 and RPM2 proline-rich motifs that are present in several other cytokine receptors (OTMeal and Y-Lee, 1993). In the region running between residues 243 and 266, the deletion of which destroys the binding of p55-R to 55.11 (see (b) and (d) above and Table 2), two of the serines and two of the threonines are followed by proline residues making them potential sites for phosphorylation by MAP kinase, CDS2 and other proline-dependent kinases (Seger and Krebs, 1995). Phosphorylation of this site in the receptor can affect its binding to the 55.11 protein.

In light of all the above mentioned with respect to protein 55.11 and its binding to p55-IC, it can be concluded that according to the present invention, a new protein has been found that binds to a distinct region upstream of the "death region" of p55-IC. Such binding may affect TNF-mediated activities distinct from the induction of cell death. The region to which 55.11 binds has previously been shown to be involved in the induction of nitric oxide synthase (Tartaglia et al., 1993) and appears to be involved in the activation of neutral sphingomeylinase by TNF (Wiegmann et al., 1994). It is thus possible that association (binding) of 55.11 with the intracellular region of p55-TNF-R (p55IC) affects or is involved in: (i) the signaling for these above-mentioned or other TNF effects, (ii) the folding or processing of the protein (as suggested by the similarity of 5.11 to a subunit of the 26S proteasome), or (iii) the regulation of the activity or expression of p55-TNF-R.

EXAMPLE 2

Self-association ability to the extracellular region of the p55 TNF receptor ( p55IO and its ability to cause cell death and other properties and activities thereof, oe a related Fas/ APO1 receptor intracellular region

As indicated in Example 1 above, it was discovered that the intracellular region of p55 TNF-R (p55IC) is capable of binding to itself, and further that fragments of p55IC, namely proteins 55.1 and 55.3, are also capable of binding to p55IC.

It has been shown that the binding of TNF to the p55 TNF-R leads to a cytocidal effect on the cells that carry this receptor. Furthermore, antibodies against the extracellular region of this receptor can themselves trigger the effect in correlation with the efficiency of the receptor cross-linking by them.

In addition, mutational studies (Tartaglia et al., (1993); Brakebusch et al., (1992)) showed that the function of p55-R depends on the integrity of its intracellular region. It was therefore proposed that the initiation of signaling for the cytocidal effect of TNF occurs as a consequence of association of two or more intracellular regions of p55-R (p55-IC), imposed by receptor aggregation. The results of the present invention provide further evidence for this by showing that the expression of the intracellular region of p55-R in the cells without the transmembrane or intracellular region triggers their death. Such free intracellular regions of p55-R have been shown to self-associate, which likely accounts for their ability to function independently of TNF. The fact that signaling by full-length p55-R is dependent on TNF stimulation has been suggested to reflect activities of the transmembrane or extracellular region of the receptor that reduce or prevent its self-association.

The ability of the intracellular region of p55-R (p55-IC) to self-associate was found to be fortunate in the attempts to clone the effector proteins that interact with this receptor (see Example 1 above). The above-mentioned "two hybrid" technique was used for this purpose. In addition to the new protein 55.11 found to associate

(binding) to p55IC, three other cloned HeLa cell cDNAs were found to contain cDNA sequences encoding parts of the intracellular region of p55-R, meaning that p55-IC is capable of self-association. Two of these clones were identical, containing an insert encoding amino acids 328-426 (designated as clone 55.1 encoding

protein fragment 55.1 of p55IC). The third contained a longer insert encoding amino acids 277-426 (designated clone 55.3 encoding protein fragment 55.3 of p55IC).

In addition, in vitro interaction between two bacterially produced chimeras of p55IC, one in which it was fused to the maltose-binding protein (MBP) and the other in which it was fused to glutathione-S-transferase (GST), was investigated. These chimeras were constructed, cloned and expressed by standard methods. After their expression, the self-interaction of

p55-R intracellular region (p55IC) by determining the interaction of the above bacterially produced chimeric proteins GST-IC55 (Mr - 51kD) and MBP-IC55 (Mr -

67 kD) with each other. Equal amounts of GST-IC55 chimera (samples from lanes 1-4 in Fig. 5) or GST alone (samples from lanes 5-8 in Fig. 5) were bound to glutathione agarose beads (Sigma) and then incubated with the same amount of MBP-IC55 fusion protein in one of the following buffer solutions: (i) buffer I (20 mM Tris-HCl, pH 7.5; 100 mM KC1, 2 mM CaCl2, 2 mM MgCl2, 5 mM DTT, 0.2% Triton X100, 0, 5 mM PMSF, 5% glycerol). This was done for the samples in tracks 1 and 5 in fig. 5. (ii) buffer I containing 5mM EDTA instead of MgCto. This was done for the samples in tracks 2 and 6 in fig. 5. (iii) buffer I containing 250 mM instead of 100 mM KCl. This was done for the samples in tracks 3 and 7 in fig. 5. (iv) buffer I containing 400 mM instead of 100 mM KCl. This was done for the samples in tracks 4 and 8 in fig. 5.

After incubation with rotation for 2 hours at 4°C, the beads were washed with the same buffers and then boiled in the SDS-PAGE buffer followed by electrophoresis by PAGE. The proteins on the gel were then Western blotted onto the nitrocellulose membrane which was then stained with polyclonal antiserum against MBP. A reproduction of these stained Western blots is shown in Fig. 5, the samples in tracks 1-8 are those indicated above.

From fig. 5, it is clear that the p55IC-MBP chimera binds to the p55IC-GST chimera (lanes 1-4) independently of divalent cations and even at a relatively high salt concentration (0.4M KCl). Thus, it was concluded that p55IC is capable of strongly self-associating.

To evaluate the functional implications of the tendency of p55-IC to self-associate, it was attempted to express p55-IC in the cytoplasm of cells sensitive to the cytocidal effect of TNF. In view of the possibility that p55-IC will be cytotoxic, it was chosen to express it in an inducible manner using the recently developed highly regulated tetracycline-controlled mammalian expression system (Gossen and Boujard, 1992). Expression of p55-IC resulted in massive cell death (Fig. 6, right panel). The dying cells showed disintegration of the cell surface as observed in the killing of cells by TNF. Transfection of the p55-IC construct into the cells in the presence of tetracycline, which reportedly reduced the expression of pHD10-3 regulated constructs as much as 10^fold, still resulted in some cell death, although it was significantly less than that observed in the absence of tetracycline ( Fig. 6, left panel). In contrast, cells transfected with a control construct containing luciferase cDNA showed no signs of cell death (results not shown).

The ability of pSS-IC to trigger cell death when expressed without transmembrane or extracellular regions of the receptor provided further evidence for the involvement of this region in signaling. Furthermore, it indicates that there are no other parts of the receptor that play a direct role in such signaling. Studies of the effects of mutations, including those studied in the present invention, on the function of p55-IC indicated that the region running between amino acid residues 326 and 407 is very critical for its function. This region shows a marked similarity to the sequence in the intracellular region of the other two receptors, which are evolutionarily related to the p55 TNF-R, namely the Fas receptor (Itoh et al., 1991); Oehm et al., 1992) which can also signal for cell death and CD40, a receptor (Stamenkovic et al., 1989) that increases cell growth; this sequence therefore seems to constitute a conservation motif that plays a kind of general role in signalling. As it does not resemble known motif characteristics for enzymatic activities, it seems plausible that it signals in an indirect manner, i.e. possibly by serving as a docking site for signaling enzymes or for proteins that transmit stimulatory signals to them. p55-IC, the Fas receptor and CD 40 can all be stimulated by antibodies against their extracellular domain. Their stimulation can be shown to correlate with the ability of antibodies to cross-link the receptor. It therefore seems as if the signaling is initiated as a consequence of interaction of two or more intracellular areas by means of aggregation of the extracellular areas. Involvement of such interaction in the initiation of signaling by these receptors was also indicated by studies (Brakebusch et al., 1992) showing that the expression of receptors rendered non-functional by mutation of their intracellular region had a "dominant negative" effect on the function of co-expressed normal receptors. Aggregation of p55-R in response to TNF was suggested to occur in a passive manner, only due to the fact that each of the TNF molecules acting as homotrimers can bind two or three receptor molecules. However, the findings of the present invention suggest that this process behaves somewhat differently.

The tendency of p55-IC to self-associate indicates that this region plays an active role in its induced aggregation. Moreover, the activity of p55-IC appears to be sufficient for the initiation of its signaling, as when expressed independently of the rest of the receptor molecule, it can trigger cell death in the absence of TNF or any other external stimuli. However, when expressed as the full-length receptor, p55-TNF-R does not signal if not stimulated by TNF. One must therefore assume that upon activation of p55-TNF-R, TNF actually overcomes some inhibitory effects that prevent spontaneous association of the intracellular regions and this inhibition occurs due to the binding of p55-IC to the rest of the receptor molecule. The inhibition may be due to the orientation provided by the intracellular region by the transmembrane or extracellular region to associate some other proteins with the receptor or may be due to limitation of the amount of receptors that can be present on the plasma membrane. This control mechanism must of course be very efficient, as according to some calculations the binding of just one TNF molecule to a cell is sufficient to trigger its death.

Spontaneous signaling independent of ligand can result in great confusion in the process controlled by the receptor. The best known example is the deregulation of the growth factor receptors. Mutations that cause them to initiate signaling spontaneously, such as those that cause them to aggregate spontaneously, play an important role in the deregulated growth of tumor cells. TNF effects, when induced in excess, are well known to contribute to the pathology of many diseases. The ability of free intracellular regions (p55IC) of p55-TNF-R to signal independently of TNF may contribute to such excessive function. It seems possible, for example, that some of the cytopathic effects of viruses and other pathogens result, not from their direct cytocidal function, but from proteolytic cleavage of the intracellular region of p55-TNF-R, and the resulting TNF-like cytotoxic effect .

To further deduce the regions of pSSIC responsible for its self-associating ability and thus its ligand-independent cellular toxicity, and also to determine whether other related members of the TNF/NGF receptor family (for example, FAS-R) also have intracellular regions of self-associating abilities and ligand-independent effects, the following detailed study was performed:

(a) General procedures and materials

(i) Two-hybrid examination and two-hybrid fi-galactosidase expression test

cDNA inserts, which coded for p55-IC and its deletion mutants, Fas-IC and various other proteins (see Table 3), were cloned by PCR, either from full-length cDNA cloned previously in applicant's laboratory, or from acquired cDNA libraries. B-galactosidase expression in yeast (SFY526 reporter strain (Bartel et al., 1993)) transformed with these

cDNA in pGBT-9 and pGAD-GH vectors (DNA binding domain (DBD) and activation domain (AD) constructs, respectively) was examined by a liquid assay (Guarente, 1983); it was also examined by a filter analysis to give quantitatively the same results (not shown). The two-hybrid assay (Fields and Song, 1989) of an acquired Gal4 AD-labeled HeLa cells cDNA library (Clontech, Palo Alto, Ca., USA) for proteins that bind to the intracellular region of p55-R (p55-IC) , was performed using the HF7c yeast reporter strain according to the manufacturer's recommendation. Positive results for isolated clones were examined by (a) prototrophy of transformed yeast for histidine when grown in the presence of 5 mM 3-aminotriazole, (b) β-galactosidase expression, (c) specific tests (interaction with SNF4 and lamin fused to Gal4 DBD).

(ii) In vitro self-association of bacterially produced p55-IC fusion proteins

Glutathione S-transferase (GST) and glutathione S-transferase-p55-IC fusion protein (GST-p55-IC) were produced as described elsewhere (Frangioni and Neel, 1993; Ausubel et al., 1994). Maltose binding protein (MBP) fusion proteins were obtained using the pMalcRI vector (New England Biolabs) and purified on an amylose resin column. The interaction of MBPP and GST fusion proteins was examined by incubating glutathione-agarose beads sequentially with GST and MBPP fusion proteins (S ug protein/20 µl beads; first incubation for 15 minutes, and the second for 2 hours, both at 4°C). Incubation with MBP fusion proteins was performed in a buffer solution containing 20 mM Tris-HCl, pH 7.5, 100 mM KC1, 2 mM CaCl2, 2 mM MgCl2, 5 mM dithiothreitol, 0.2% Triton X100, 0.5 mM phenyl-methyl- sulfonyl fluoride and 5% (v/v) glycerol, or when indicated, in the same buffer containing 0.4 M KCl, or 5 mM EDTA instead of MgCl. Association of MBP fusion proteins was examined by SDS polyacrylamide gel electrophoresis (10% acrylamide) of those proteins associated with glutathione-agarose beads, followed by Westem blotting. The blots were tested with rabbit antiserum against MBP (produced in the applicant's laboratory) and with pepperot peroxidase-linked goat anti-rabbit immunoglobulin.

(tii) Induced expression in HeLa cells of p55-R and fragments thereof

HeLa cells expressing the tetracycline-controlled transactivator developed by Gossen and Bujard (HtTA-1 clone (Gossen and Bujard, 1992)) were grown in Bulbecco's modified Eagle's medium containing 10% fetal calf serum, 100 u/ml penicillin, 100 ug/ml streptomycin and 0.5 mg/ml neomycin. cDNA inserts encoding p55-R or parts thereof were introduced into a tetracycline-controlled expression vector (pUHD 10-3, which was provided by H. Bujard). The cells were transformed with the expression construct (5 µg DNA/6 cm plate) by the calcium phosphate precipitation method (Ausubel et al., 1994). Effects of transient expression of transfected proteins were examined at the indicated times after transfection in the presence or absence of tetracycline (1 µg/ml). Clones of cells stably transfected with the human p55-IC cDNA in the pUHD 10-3 vector were stabilized by transfecting the cDNA into HtTA-1 cells in the presence of tetracycline together with a plasmid conferring resistance to hygromycin, followed by selection for clones which were resistant to hygromycin (200 ug/ml). Expression of cDNA was achieved by removal of tetracycline which was otherwise maintained constant in the cell growth medium.

(iv) Measurement of TNF-like effects, triggering of induced expression of p55-R and fragments thereof

Effects of induced expression of the receptor and TNF on cell viability were examined by the neutral-red uptake method (Wallach, 1984). Induction of IL-8 gene expression was examined by Northem analysis. RNA was isolated using TRI REAGENT (Molecular Research Center, Inc.), denatured in formaldehyde/formamide buffer, electrophoresed through an agarose/formaldehyde gel and blotted onto a GeneScreen Plus membrane (Du Pont) in lOxSSPE buffer using standard techniques. Filters were hybridized with an IL-8 cDNA probe (Matsushima et al., 1988), nucleotides 1-392), radiolabeled by random-prime kit (Boehringer Mannheim Biochemica, Mannheim, Germany) and washed stringently according to the manufacturer's protocol. Autoradiography was performed for 1-2 days.

(v) Examination of TNF receptor expression

TNF receptor expression in samples of lxl0<6> cells was investigated by measuring binding of TNF, labeled with <12>^i by the chloramine-T method, as previously described (Holtmann and Wallach, 1987). It was also examined by ELISA, performed as described for the quantification of soluble TNF receptors (Aderka et al., 1991) except for the use of RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS and 1 mM EDTA) to lyse the cells (70 µl/10<6> cells) and to dilute the test samples. The soluble form of p55-R, purified from urine, served as a standard.

(b) Mutational analysis of the intracellular region of p55-R (p55-IC) to determine the regions of p55-IC involved in its self-association

As indicated above, p55-IC can self-associate and trigger cytotoxic effects on cells, and there are parts of p55-IC that are themselves capable of binding to full-length p55-IC. In particular, one of the parts of p55-IC (designated as protein fragment 55.1 in Example 1 above) was identified as capable of strong binding to full-length p55-IC, this part was sequenced and was observed to contain amino acid residues 328-426 of p55 -TNF-R, which is within p55-IC. It was further discovered (see below) that the above portion, protein fragment 55.1, is itself capable of self-association and of triggering cytotoxic effects on cells. Thus, this part of p55-IC has been called the "dead region" and is located in the region between amino acid residues 328-426 of the human p55-R, most likely consisting of the amino acid residues between about residue 328 and 414 thereof.

The fact that the "dead zone" of p55-IC self-associates was found by serendipity. When examining a HeLa cell cDNA library by the two-hybrid technique (see example 1 above) for proteins that bind to the intracellular region of this receptor, several clones were found among cDNAs whose products bound specifically to the intracellular region of the GAL4 DBD fusion protein (eg 55.1 and 55.3) which themselves encode parts of the p55-R intracellular region (p55-IC, marked with an asterisk in Table 3).

The application of the two-hybrid test to evaluate the degree of specificity in the self-association of p55-IC and to define more precisely the region involved led to the following findings (Table 3): (a) The self-association of p55-IC is given to a region within the "dead area". Its N terminus is located between residues 328 and 344 and its C terminus is close to residue 404, slightly upstream of the reported C terminus of this region (residue 414). (b) Deletion of the membrane-proximal part of p55-IC upstream of the "death region" increased self-association, suggesting that this region has an inhibitory effect on association, (c) Mouse p55-IC self-associates, and also associates with the "dead zone" of human p55-R. (d) Examination of the self-association of the intracellular regions of the other three receptors of the TNF/NGF receptor family: Fas/APO1 (FAS-R), CD40 (Fields and Song, 1989) and the p75 TNF receptor (Smith et al., 1990), showed that Fas-IC which signals for cell death by a sequence motif related to the p55-R "death region, self-associates, and associates to some extent with p55-IC. However, CD40-IC which provides growth-stimulatory signals (although it also contains a sequence corresponding to the "death region") and p75-IC, which has no structural similarity to p55-IC, does not self-associate nor bind to p55-IC or Fas-IC.

Table 3 above shows that the quantitative evaluation of the interaction of the Gal4 hybrid constructs includes the following proteins: the intracellular region of

human p55-R and its various deletion mutants (residues numbered in (Loetscher et al., 1990)); the intracellular regions of mouse p55-R (residues 334-454, numbered as in (Goodwin et al., 1991)); mouse Fas/APO1 (Fas-IC, 166-306, numbered as in (Watanabe-Fukanaga et al., 1992)); human CD40 (CD40-IC, 216-277, numbered as in (Stamenkovic et al., 1989)); and human p75 TNF receptor (p75-IC, 287-461, numbered as in (Smith et al., 1990)). SNF1 and SNF4 were used as positive controls for association (Fields and Song, 1989), and lamin as a negative control (Bartel et al., 1993). Proteins encoded by the Gal4 DBD constructs (pGPT9) are listed vertically; they encoded by Gal4 AD constructs (pGAD-GH) horizontally. The two deletion mutants indicated by asterisks were cloned in a two-hybrid screen of a HeLa cell cDNA library (Clontech, Palo Alto, Ca., USA) using p55-IC cloned in pGBT9 as "bait". In that screen, four of the 4xl0<6> examined cDNA clones were positive. Three of these clones were found to correspond to portions of human p55-R cDNA (two were identical, encoding residues 328-426 and one encoding residues 277-426). The fourth was found to encode an unknown protein. The 6-galactosidase expression data are averages of analyzes of two independent transformants and are presented as amount of 6-galactosidase product; (a unit of activity is defined as OD420 times 10-<*> divided by ODgQO of the yeast culture and reaction time in minutes). The detection limit of the assay was 0.05 units. Variation between duplicate samples was in all cases less than 25% of the mean (not tested). ;An in vitro test of the interaction of a p55-IC-glutathione-S-transferase (GST) bacterial fusion protein with a p55-IC-maltose-binding protein (MBP) fusion protein confirmed that p55-R self-associates and ruled out the involvement of yeast proteins in this the association (see above). The association was not affected by increased salt concentration or by EDTA (see above). ;To evaluate the functional implications of death site self-association, the manner in which induced expression of p55-R or parts thereof affected cells sensitive to TNF cytotoxicity was investigated. The results of this analysis are shown in fig. 7 which indicates the ligand-independent triggering of a cytocidal effect in HeLa cells transfected with p55-R, its intracellular region (p55-IC) or parts thereof (including the "death region"). In fig. 7 schematically shows the different DNA molecules that code for the different types of TNF receptors included in the vectors with which HeLa cells were transfected (far left in fig. 7); and the expression (left and middle curves) and viability (right curve) in HeLa cells transiently expressing the different full-length p55-R (p55-R), p55-IC or parts of p55-IC, or as a control, luciferase (LUC) (each of which is indicated on the far left of Fig. 7), using a tetracycline-controlled expression vector. The open bars (left, middle and right) represent cells transfected in the presence of tetracycline (1 µg/ml), which inhibits expression, and the filled columns (left, middle and right) represent cells transfected in the absence of tetracycline. TNF receptor expression was examined 20 hours after transfection, both by ELISA using antibodies against the receptor's extracellular region (see schematic illustration on the left side in Fig. 7) and by determining the binding of radiolabeled TNF by the cells (middle). The cytocidal effect of the transfected proteins was examined 48 hours after transfection. The data shown are from one of three experiments with qualitatively similar results, in which each construct was tested in duplicate. ND - not determined. ;From fig. 7, it is thus clear that using an expression vector that allows strictly controlled expression of transfected cDNA by a tetracycline-regulated transactivator (Crossen and Bujard, 1992) resulted in a small increase of p55-R expression in HeLa cells upon expression of transiently transfected cDNA for full- length receptor, in relatively large cell death. An even greater cytotoxicity was observed upon expression of only p55-IC. Significant cytotoxicity was also observed upon expression of only parts of p55-IC comprising mainly the "death region" (residues 328-426) in HeLa cells. On the other hand, expression of parts of p55-IC that lacked the "death region" or contained only parts thereof (or expression of the luciferase gene, used as an irrelevant control) had no effect on cell viabilities. The cytotoxicity of p55-IC was further confirmed using cells stably transformed with its cDNA; these cells continued to grow when p55-IC expression was not induced, but died when p55-IC was expressed (see above). ;( c) Other effects of the intracellular region of p55-TNF-R ;To investigate whether other activities of TNF are triggered by self-association of the intracellular region, including hence the "death region", the effect of increasing the expression of full -length receptor (p55-R) and the expression of the intracellular region of the receptor (p55-IC) on the transcription of interleukin 8 (IL-8), which is known to be activated by TNF (Matsushima et al., 1988) investigated . The results are shown in fig. 8 showing the ligand-independent induction of IL-8 gene expression by HeLa cells transfected with p55-R or p55-IC using a tetracycline-controlled construct (see also "General Procedures and Materials" and Example 1 above). In panel A in fig. 8 shows a reproduction of a Northern blot showing the Northern blot analysis (see "General Procedures and Materials" above) of RNA (7 µg/lane) extracted from HeLa (HTta-1) cells, untreated ("control" ) or treated ("TNF") with TNF (500 ug/ml for 4 h), or HTta-1 cells 24 h after transfection (in the presence or absence of tetracycline) with p55-IC ("p55-IC"), p55 -R ("p55-R"), or luciferase ("Lue") cDNA. In panel B of fig. 8 shows a reproduction of a Northern blot showing methylene blue staining of 18S rRNA in each of the samples shown in panel A in fig. 8. As is clear from fig. 8, thus transfection of HeLa cells with a tetracycline-controlled construct encoding p55-R cDNA induced IL8 transcription. Even a stronger induction was observed in cells transfected with cDNA for p55-IC. In both cases, induction occurred only when tetracycline was excluded from the cell culture medium, indicating that it occurs as a consequence of expression of the transfected p55-R or p55-IC. Transfection with luciferase cDNA as a control had no effect on IL-8 transcription. Accordingly, from the above results (Fig. 8), it appears that a small increase in p55-R expression or even expression of only the intracellular region (p55-IC) thereof is sufficient to trigger a ligand (TNF)-independent manner, cytotoxicity and also other effects including that of an increase in the expression of the IL-8 gene in cells. The release of these effects is likely due to the self-association of the intracellular region of p55-R (p55-IC). As indicated above, it appears that after self-association of p55-IC the "death zone" is mainly responsible for signaling the induction of the intracellular processes leading to the triggering of cytotoxicity in the cells, while the other effects, such as signaling leading to the induction of IL-8 gene expression, probably due to other regions of p55-IC, following its self-association. It is therefore possible that different regions of p55-IC are responsible for different TNF-induced effects (eg cytotoxicity, BL-8 induction) inside the cells, where these effects are a consequence of the intracellular signaling after self-association of p55- IC. The fact that p55-IC can induce, in a ligand (TNF)-independent manner, the triggering of other intracellular effects, for example IL-8 induction, means that p55-IC or specific parts thereof can be used as highly specific tools to produce effects in cells or tissues that are desired to be treated, without the need to treat such cells or tissues with TNF. For many pathological conditions (eg, malignancy), treatment with TNF, especially at high doses, can lead to undesirable side effects due to the number of intracellular effects induced systemically by TNF after its binding to its receptors. By means of the discovery according to the present invention that p55-IC can mimic specific other TNF-induced effects (besides cytotoxicity) for example IL-8 induction, the way is opened for the introduction in a cell- or tissue-specific way, of p55-IC or specific parts thereof, which will be able to signal for the induction of specifically desired intracellular effects, for example IL-8 induction, thereby overcoming the systemic side effects that are often observed with TNF treatment. ;( d) Ligand-independent triggering of cytocidal effects in HeLa cells at the intracellular areas and "death areas" thereof by p55 TNF-R oe FAS-R (Fas/APO!) ;What concerns the cytotoxic activity of the intracellular areas of p55 TNF- R and FAS-R (pttIC and FAS-IC) it has now been further developed that both p55IC, its "dead region" (p55DD) and FAS-IC are capable of a ligand-independent triggering of a cytocidal effect in HeLa cells. In this study, HeLa cells were transfected with expression vectors containing various constructs of either full-length p55-TNF-R, parts thereof including p55IC and p55DD or FAS-IC. In one set of experiments, HeLa cells were co-transfected with constructs containing p55 TNF-R (p55-R) and FAS-IC (for details of the constructs, their preparation, etc., see above). The results of this study are shown in fig. 9 (A and B), where in both fig. 9A and B the constructs used for transfection of HeLa cells are shown schematically in the panels on the left; the results of TNF or FAS receptor expression are shown graphically in the two middle panels (second and third panels from the left), and the results for transfected cell survival are shown graphically in the right panel. In fig. 9A shows the results of transfected HeLa cells transiently expressing full-length p55-R, p55-IC or parts thereof, or as a control, luciferase (LUC), in all cases using a tetracycline-controlled expression vector. In fig. 9B shows the results of transfected HeLa cells transiently expressing FAS-IC alone or together with p55-R using a tetracycline-controlled expression vector. In the graphic representation in fig. 9A and B, open bars represent cells transfected in the presence of tetracycline (1 µg/ml), which inhibits expression, and the filled bars represent cells transfected in the absence of tetracycline. TNF receptor expression was measured 20 hours after transfection both by ELISA using antibodies against the extracellular region of the receptor (see panels on the left), and by determining the binding of radiolabeled TNF to the cells (middle panels). The cytocidal effect of the transfected proteins was measured 48 hours after transfection. The data shown are from one of three experiments with qualitatively similar results where each construct is tested in duplicate. The designation "ND" in fig. 9A and B mean not determined. From the results shown in fig. 9A and B, it is clear that expression of p55IC alone results in even greater cytotoxicity. Significant cytotoxicity can also occur upon expression of only the death domain (p55DD). In contrast, expression of parts of p55IC lacking the death domain or containing only parts of it had no effect on cell survival. Expression of FAS-IC did not result in significant cytotoxicity, although it significantly increased the cytotoxicity of co-expressed p55-R. ;EXAMPLE 3: ;Additional proteins capable of binding to the intracellular regions of p55 TNF-R or FAS-R ;Using the same approach and technology as set forth in Example 1 above, three additional proteins were isolated and identified to to be able to bind to p55IC or ;FAS-IC. In fig. 10-12 is shown schematically the partial and preliminary nucleotide sequence of cDNA clones, named F2, FO and DD 11. Clones F2 and F9 were isolated by screening murine (mouse) embryonic library using murine FAS-IC as "ate ". In fig. 10 schematically shows the partial nucleotide sequence from the F2 cDNA that had been sequenced. In fig. 11 schematically shows the partial nucleotide sequence of 1724 bases from the F9 cDNA that had been sequenced. Analysis of the binding capacity of the protein encoded by clones F2 and F9 (F2 and F9 respectively) has shown that: (a) F2 interacts strongly with human p55IC and p55DD and with murine FAS-IC, while it interacts weakly with non-relevant (control) proteins SNF1 and lamin as well as the relevant protein, human FAS-IC. (b) F9 interacts strongly with human p55-IC and murine FAS-IC, while it interacts weakly with human FAS-IC (relevant protein) and irrelevant proteins SNF1 and lamin. (c) Neither F2 nor F9 interacted at all with human p75IC, pGBT9 (empty bait vector) or human CD-40. Furthermore, from "Gene Bank" and "Protein Bank" searches, it was revealed that F2 and F9 represent new proteins. ;F2 and F9 thus represent new proteins that had binding specificity for both FAS-IC and p55IC. Clone DD11 was isolated by screening a human HeLa library using human p55DD as "bait". In fig. 12 schematically shows the partial nucleotide sequence of 425 bases from the DD11 cDNA that had been sequenced. The DD11 clone has an approximate length of 800 nucleotides. The full length of the transcript is approximately 1.2 kb, where the transcript has been sampled using the clone. Analysis of the binding ability of the protein encoded by clone DD11 has shown that DD11 interacts strongly with p55DD (amino acids 326-414) (see Fig. 9) and does not interact with deletion mutants in this region, for example amino acids 326-404. DD11 also interacts with mouse and human FAS-IC and to some extent with lamin. DD11 does not interact at all with SNF1 or with pGBT9 (empty bait vector). DD11 was also not found in the "Gene Bank" and "Protein Bank" databases. DD11 thus represents a p55 IC (p55DD) and FAS-IC specific binding protein. ;EXAMPLE 4 ;Construction of soluble dimeric TNF receptors ;Based on the findings set forth in Example 2 above that the intracellular region of p55-R (p55-IC) and part thereof ("death region") and that the intracellular region of Fas/APOl and a part thereof (also called the "death zone") corresponding to the p55-IC "death zone" are capable of self-association, it is possible to construct new TNF receptors capable of self-association (aggregation ) and which are soluble. Such TNF receptors will be fusion proteins having substantially all of the extracellular region of p55-R fused with substantially all of the intracellular regions or "dead regions" thereof of p55-R or Fas/APO1. Such fusion constructs will thus be without the transmembrane region of p55-R (or FAS/APO1) and thus be soluble. Furthermore, by the self-association of the intracellular regions or "dead regions", these fusion constructs will be capable of oligomerization to give at least dimers (and possibly also higher order multimers) of p55-R. Accordingly, such dimeric TNF receptors (p55-R) will be capable of binding to at least two TNF monomers of naturally occurring TNF homotrimers to give a soluble TNF receptor that binds more strongly to its ligand (homotrimer TNF). ;Therefore, at least four types of p55 TNF receptor fusion proteins will be constructed, each of which will be capable of oligomerization and will be soluble: (i) A fusion product between the extracellular region of pSS-R (ECSS) and the intracellular region of p55 -R (p55-IC); (ii) A fusion product between EC55 and the “death domain” of p55-IC (DD55); (iii) A fusion product between EC55 and the intracellular domain of Fas/APO1 (ICFAS); and; (iv) A fusion product between EC55 and the "death domain" of ICFAS (DDFAS). ;In each of the above fusion proteins, TNF monomer binding capacity is conferred by the EC55 portion while oligomerization (or at least the dimerization) of each type of fusion protein is conferred by its "tail" region which is any of the p55IC, DD55, ICFAS or DDFAS portions . For the construction of the above fusion proteins, standard techniques of recombinant DNA technology will be used which are now well established in the art (see for example Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor , N.Y.), any suitable bacterial, bacteriophage or animal viral expression vector (clone carrier or plasmid designed for expression of the inserted DNA) is used in which is inserted in one or more of the steps DNA encoding EC55 and one of " the tails" which is p55. IC, DD55, ICFAS or DDFAS. The thus inserted DNA encoding each of the fusion proteins will be placed under the control of various expression controlling sequences of the clone carrier or plasmid such as promoters, ribozyme binding sites, transcription factor binding sites, etc. These expression controlling sequences will be chosen depending on the type of expression vector chosen and thus the type of host cell (eukaryotic or prokaryotic) in which it is desirable to express the fusion proteins. Preferred host cells (and thus expression vectors) are eukaryotic, especially mammalian. ;The DNA molecule that codes for each of the above-mentioned fusion proteins will be prepared and inserted into the expression vector by the following procedure: (a) First, a set of oligonucleotides for use in PCR will be constructed by standard means, where the oligonucleotides are: ;1) ACC ATG GGC CTC TCC ACC GTG (EC55, sense) ;2 ACGC GTC GAC TGT GGT GCC TA GTC CTC (EC55, antisense) ;3) ACGC GTC GAC CGC TAC CAA CGG TGG AAG (IC55, sense) ;4) TCATCT GAG AAG ACTGGG(IC55, antisense) ;5) ACGC GTC CAG AAG AGA AAG GAAA GTA CAG (IC FAS, sense) ;6) CTA GAC CAA GCT TTG GAT (IC FAS, antisense) ;7) ACGC GTC GAC CCC GCG ACG CTG TAC GCC (DD55, sens) ;8) ACGC GTC GAC GAT GTT GAC TTG AGT AAA (DD FAS, sens) ;(b) Plasmids containing the cloned full-length p55-R and Fas/APOl receptors that the applicants have in the laboratory ( see also parallel EP56892S and examples 1-3 above) will be subjected to the following manipulations to provide the DNA fragments encoding each of the fusion proteins, if the DNA fragments then is ligated into the expression vectors selected above: (i) To prepare the DNA fragment encoding EC55 which is a component of all 4 fusion proteins, PCR is performed on a plasmid carrying the cDNA of human p55 using the oligonucleotides No. 1 and 2 above (size of fragment 640 bp). (ii) To obtain a fusion product EC55-IC55, PCR is performed on a plasmid carrying cDNA for human p55 using oligonucleotides No. 3 and 4 to obtain a DNA fragment encoding ICSS (size 677 bp) which then is mixed with ECSS cut at Sal I and ligated by buttend ligation into any mammalian cell expression vector under the control of an appropriate promoter. The orientation of the inserted EC55--IC55 in the vector is verified for restriction cutting and by sequencing. (iii) To obtain a fusion product EC55-IC FAS, IC FAS is produced by PCR on a plasmid with cDNA for FAS using oligonucleotides No. 5 and 6 to obtain a fragment (size 448 bp) which is then cut with Sal I and mixed with ECSS cut at SalI and then buttdelegated into a mammalian expression vector under the control of an appropriate promoter. The orientation of the inserted EC55--IC FAS in the vector is verified by restriction digestion and by sequencing. (iv) To obtain a fusion product EC55--DD55, a DNA fragment is produced with the DD55 sequence by PCR in cDNA for human p55 using oligonucleotides No. 7 and 4. The product with a size of 314 bp is cut with SalI and mixed with EC55 cut with SalI and then buttdelegated into the mammalian expression vector. Orientation of the inserted EC55-DD55 in the vector is verified by restriction cutting and sequencing. (v) To obtain a fusion product EC55-DD FAS, a DNA fragment with DD FAS is produced by PCR on cDNA for FAS using oligonucleotides No. 6 and 8. The product with a size of 332 bp is cut with SalI and mixed with EC55 cut at Sali and then buttdelegated into the mammalian expression vector. The orientation of EC55--FAS is then verified by restriction cutting and sequencing. ;Once the above expression vectors have been constructed, they will then be introduced by standard methods into appropriate mammalian cells (eg, Chinese Hamster Ovary (CHO) or Monkey Kidney (COS) cells) for expression purposes. The thus expressed fusion proteins will then be purified by standard methods (see parallel applications EP308378; EP398327 and EP568925). The purified fusion proteins will then be analyzed for their ability to oligomerize (and the degree thereof, ie whether they form dimers or higher order multimers) and for their ability to bind TNF (and the affinity or strength of binding thereof). ;EXAMPLE 5 ;Construction of soluble dimeric Fas/APOl receptors ;In a similar manner as indicated in example 4 above, it is possible to produce the following four types of Fas/APOl fusion products, each of which will be capable of oligomerization and will be soluble: ;(i) Fusion product between the extracellular region of Fas/APO1 (EC FAS) and the intracellular region of p55-IC; (ii) Fusion product between EC FAS and the “dead domain” of p55-IC (DD55); (iii) Fusion product between EC FAS and the intracellular domain of Fas/APO1 (IC FAS); and ;(iv) Fusion product between EC FAS and the "dead area" of IC FAS (DD FAS). ;In each of the above fusion proteins, the FAS ligand-binding ability is conferred by the EC FAS portion, while the oligomerization (or at least dimerization) of each type of fusion protein is conferred by its "tail" region which is any of p55-IC, DD55, IC FAS or DD FAS parts. ;The construction of the DNA fragments encoding the above fusion proteins and the expression vectors containing them will be as indicated in Example 4, except that different appropriate oligonucleotides (not shown) will be used to prepare the EC FAS fragment to be ligated to any of the above indicated "tail" regions. Next, the expression vectors will be introduced into the appropriate host cells and the resulting expressed fusion proteins will be purified and tested for their ability to oligomerize (or the degree thereof, ie whether they form dimers or higher order dimers) and for their ability to bind the FAS ligand (and the affinity or degree of binding thereof). ;EXAMPLE 6 ;Construction of soluble oligomeric "mixed" TNF/ FAS receptors ;To prepare oligomeric receptors that have "mixed" affinity, i.e. affinity for both TNF and the FAS-R ligand, the above mentioned (examples 4 and 5) the fusion products be used in the following procedure: i) Preparation of a fusion product as set forth in Example 4 containing the extracellular region of a TNF-R (p75 TNF-R or p55 TNF-R) fused to any of: p55 IC , FAS-IC, p55 DD or FAS DD; ;ii) Obtaining a fusion product as set forth in Example 5 containing the extracellular region of Fas-R fused to any of: p55 IC, FAS-IC, p55 DD or FAS-DD; and ;iii) Mixing any of the fusion products of i) with any of the fusion products of ii) to provide a new dimeric (or higher order oligomeric) receptor having both the extracellular regions of a TNF-R and FAS-Rs that are fused at their -IC or -DD regions. In the above procedure, the fusion products of i) and ii) can be obtained separately, namely for their purification from transformed cells in which they are produced, and then mixed in vitro to obtain the mixed affinity receptors. Alternatively, the host cells can be co-transfected with vectors carrying sequences encoding both types of fusion products, in which case the mixed affinity receptors can be obtained directly from the co-transfected cells. The actual oligomerization of the fusion products into oligomeric receptors can occur in the cells or during or after the purification procedure to obtain fusion products expressed in the cells. To specifically select for the mixed affinity receptors any standard method can be used, for example affinity chromatography procedures in which antibodies against TNF-R and FAS-R extracellular regions are used in sequential chromatographic steps to select for those receptors that have both types extracellular areas. ;REFERENCES ;Aderka, D., Englemann, H., Hornik, V., Skornick, Y., Levo, Y., Wallach, D. and Kushtai, G. ;(1991) Cancer Res. 51 5602-5607. ;Båens et al. (1993) Genomics 16:214-218. ;Barinaga, M. (1993} Science 2621512-4. ;Bartel, P.L., Chien, C.T., Sternglanz, R. and Fields, S. (1993) Bio Techniques J4, 920-924. Berger, J.. Hauber, J ., Hauber, R., Geiger, R. and Cullen, B.R. (1988) Gene 64 1-10.;Beutler, B. and Cerami, C. (1987) NEJM, 3J£:379-385.;Boldin, M.P. et al. (1995) J. Biol. Chem. 270,337-341. ; Bollon, D. P. et al. (1980) J. Clin. Hematol. Oncol. JO, 39-48. ; Botstein, D. et al. (1982 ) Miami-Wint. Smyp. 19, 265-274. ;Brakebusch, C. et al. (1992) EMBO J., U:943-950. ;Broach, J.R. (1981) in : The Molecular Biolog<y> of the Yeast Saccharom<y>ces : Life Cycle and ;Inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 445-470. ;Broach, J.R. (1982) Cell 28, 203-204. ;Brockhaus, M. et al. (1990) Proe Nat Acad Sci USA, 87:3127-3131. ;Cantor, G. H. et al (1993) Proe Nat Acad Sci USA 20:10932-6. ; Chater, K. F. et al. . (1986) in : Sixth International Symposium on Actinomvcetales Biology. ;Akademiai Kaido, Budapest, Hungary, 45-54. Chen, C.J. e etc. (1992) Ann N.Y. Acad. Pollock. 660:271-3. ;Cheng, J., Zhou, T., Liu, C. Shapiro, J.P. Brauer, M., Kiefer, M.C., Barr, P.J. and Mountz, J.D. ;(1994) Science 263. 1759-1762. Crisell, P. et al., (1993) Nucleic Acids Res. (England) 2_L(22):5251-5. ;Crowe, P~. D. et al., (1994) Science, 264:707-709. ;Current protocols in molecular biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., ;Seidman, J.G., Smith, J.A., Struhl, K., Albright, L.M., Coen, D.M. & Varki, A. , eds.), (1994) pp. 8.1.1-8.1.6 and 16.7-16.7.8, Greene Publishing Associates, Inc. and Wiley &;Sons, Inc., New York. DeMartino, G.N. Moomaw, C.R., Zagnitko, O.P., Proske, R.J., Chu-Ping, M., Afendis, S.J., ;Swaffield, J.C. and Slaughter, CA. (1994) J. Biol. Chem. 269, 20878-20884. Dirks, W., Wirth, M. and Hauser, H. (1993) Gene 128, 247-249. ;Endo, R, Akahoshi, T., Nishimura, A., Tonegawa, M., Takagishi, K.t Kashiwazaki, Matsushima, ;K. and Kondo, H. (1994) Clin. Exp. Immunol. 26, 31-35. Engelmann, H. et al. (1990) J. Biol. Chem., 265:1531-1536. ;Ferrick, M.R., Thurau, S.R., Oppenheim, M.H., Herbort, C.P., Ni, M., Zachariae, C.P., ;Matsushima, K. and Chan, CC. (1991) Invest. Ophthalmol. View. Pollock. 32, 1534-1539. Fields, S. and Song, 0. (1989) Nature, 540:245-246. ;Frangioni, IV. and Neel, B.G. (1993) Anal. Biochem. 210. 179-187. Glick, B.R. (1987) J. Ind. Microbiol. 1 277-282. ;Goodwin, R.G., Anderson, D., Jerzy, R., David, T., Brannan, C.I., Copeland, N.G. Jenkins, N.A. and Smith, CA. (1991) Mol. Cell Biol. IL 3020-3026. ;Gossen, M. and Boujard, H. (1992) Proe. Nati. Acad. Pollock. USA, 89:5547-5551. ;Gryczan, T. (19821 The Molecular Biolog<y> of the Bacilli. Academic Press, N.Y. 307-329. Guarente, L. (1983) in Methods Enzymol. 101. 181-191. ;Harada, A., Sekido, N., Kuno, A., Akiyama, M., Kasahara, T., Nakanishi, I., Mukaid, and ;Matsushima ;K. (1993) Int. Immunol. i 681-690. ;Heller, R. A. et al. (1990) Proe. Nat. Acad. Sci. USA, 87:6151-6155.; Hohmann, H.-P. et al. (1989) J. Biol. Chem., 26* 14927-14934.

Holtmann, H. and Wallach, D. (1987) J. Immunol. m 1161-1167.

Itoh, N. et al. (1991) Cell 66:233.

Itoh, N. and Nagata, S. (1993) J. Biol. Chem. 268, 10932-7.

Izaki, K. (1978) Jpn. J. Bacteriol. 33, 729-742.

John, J.F. et al. (1986) Rev., Infect. Haze. 8, 693-704.

Joseph, S. and Burke, J.M. (1993) J. Biol. Chem. 2^8:24515-8.

Kendall, K.J. et al (1987) J. Bacteriol 169, 4177-4183.

Khan, A.S. et al. (1992) Nature Genets, 2: 180-185.

Koizumi, M. et al. (1993) Biol. Pharm. Bull (Japan) 16 (9):879-83.

Kunkel, T.A. (1994) in : Current protocols in molecular biology, pp. 8.1.1-8.1.6 (Ausubel, F.M.

et al., eds.) Greene Publishing Associates, Inc. and Wiley & Sons, Inc., New York. Loetscher, H., Pan, Y-CE., Lahm, H.-W, Gentz, R., Brockhaud, M., Tabuchi, H. and Lesslauer,

W. (1990) Cell, 61:351-359.

Maniatis, T. et al. (1982) Molecular Cloning : A Laboratory Manual. Cold Spring Harbor

Laboratory, Cold Spring Harbor.

Maniatis, T. (1980) in : Cell Biology : A Comprehensive Treatise Vol. 3 : Gene Expression.

Academic Press, N.Y. 563-608.

Matsushima, K., Morishita, K., Yoshimura, T., Lavu, S., Kobayashi, Y., Lew, W., Appella, E.,

Kung, H.F., Leonard, E.J. and Oppenheim, J.J. (1988) J. Exp. Med. 167, 1883-1893. Nophar, Y. et al. (1990) EMBO J., £:3269-3278.

Oehm, A. et al. (1992) J. Biol. Chem. 261:10709.

Ogasawara, I, Watanabe-Fukunaga, R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y.,

Itoh, N., Suda, T, and Nagata, S. (1993) Nature 364, 806-809.

Okayama, H. (1983) Mol. Cell Biol. 3, 280.

CWeal, K.D. and Yu-Lee, L.Y. (1993) Lymphokine Cytokine Res. 12,309-312.

Piquet, P.F. etc. (1987) J. Exp. Med., 166:1280-89.

Realini, C, Rogers, S.W. and Rechsteiner, M. (1994) FEBS Lett 348, 109-113.

Rechsteiner, M., Hoffman, L. and Dubiel, W. (1993) J. Biol. Chem. 268, 6065-6068.

Sambrook et al (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory

Pressure. Cold Spring Harbor, NY.

Schall, T.J. et al. (1990) Cell, 6J.:361-370.

Schwalb et al. (1993) J. Biol. Chem. 261(14) :9949-54.

Seger, R. and Krebs, E.G. (1995) FASEB J. in press.

Sekido, N., Mujaida, N., Harada, A., Nakanishi, I., Watanabe, Y., Matsushima, K. (1993) Nature

365. 654-657.

Shimayama, T. et al.” [ 1993) Nucleic Acids Symp. Looking. 29_: 177-8

Shore, S.K. et al. (1993) Oncogene 8:3183-8.

Smith, C. A., David, T., Anderson, D., Solam, L., Beckmann, M. P., Jerzy, R., Dower, S. K.,

Cosman, D. and Goodwin, R.G. (1990) Science, 248:1019-1023.

Smith, D.B. and Corcoran, L.M. (1994) in : Current protocols in molecular biology, pp. 16.7.1-16.7.8 (Ausubel, F.M. et al., eds.) Greene Publishing Associates, Inc. and Wiley & Sons,

Inc. New York.

Song, H.Y. et al. (1994) J. Biol. Chem. 269, 22492-22495.

Stamenkovic, I., Clark, E.A. and Seed, B. (1989) Embo J. 8:1403-1410.

Tartaglia, L.A., Ayres, T.M., Wong, G.H. and Goeddel, D.V. (1993) Cell, 74:845-853.

Tracey, J.T. et al. (1987) Nature, 320:662-664.

Wallach, D. (1984) J. Immunol. J32, 2464-9.

Wallach, D. (1986) in Interferon 7 (Ion Gresser, ed.), pp. 83-122, Academic Press, London Wallach, D. et al. (1994) Cytokine & 556.

Watånabe-Fukanaga, R., Brannan, CL, Itoh, N., Yonehara, S., Copeland, N.G., Jenkins, N.A.

and Nagata, S. (1992) J. Immunol. J4S, 1274-1279.

Watanabe-Fukunaga, R. et al. (1992) Nature, 356, 314-317.

Wiegmann, K., Schutze, S., Machleidt, T., Witte, D. and Kronke, M. (1994) Cell 78, 1005-1015. WUks.. A.F. et al. (1989) Proe. Nati. Acad. Pollock. United States, £6:1603-1607.

Zhao, J.J. and Pick, L. (1993) Nature (England) 3J55_:448-51.

Claims (16)

1. DNA sequence encoding a protein capable of binding to an intracellular domain of a receptor belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor superfamily, preferably selected from the group consisting of p55 TNF-R, p75 TNF-R and FAS- the ligand receptor, said DNA sequence being selected from the group consisting of: (a) DNA sequences comprising a nucleotide sequence encoding the protein 55.1 with the amino acid sequence corresponding to amino acid residue 328 to residue 426 in the p55 TNF-R amino acid sequence; (b) DNA sequences comprising a nucleotide sequence encoding proE6n 55.3 with the amino acid sequence corresponding to amino acid residue 277 to residue 426 of the p55 TNF-R amino acid sequence; (c) DNA sequences comprising a nucleotide sequence encoding the protein 55.11 having the amino acid sequence given in Figure 4; (d) DNA sequences encoding a protein selected from the group consisting of the herein designated proteins 75.3 and 75.16 comprising the nucleotide sequence given in Figure 1(b) and Figure 1(c), respectively; (e) DNA sequences encoding any of the proteins F2, F9 and DD11, comprising a DNA sequence selected from the group consisting of the nucleotide sequence given in the first and second parts of Figure 10, respectively, the nucleotide sequence given in the first, second and third part of Figure 11 and the nucleotide sequence given in Figure 12; (f) DNA sequences capable of hybridizing to a DNA sequence according to any of items (a) to (e) under moderately stringent conditions, and (g) DNA sequences which are degenerate as a result of the the genetic code relative to the DNA sequences according to any one of items (a) to (f). 2. Vector comprising a DNA sequence according to claim 1. 3. Transformed eukaryotic or prokaryotic host cell containing a vector according to claim 2. 4. Process for producing the protein, analogs or derivatives capable of binding to an intracellular domain of a receptor belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor superfamily, comprising culturing the transformed host cell according to claim 3 under conditions suitable for expression of said protein, analogues or derivatives, causing post-translational modification of said protein as necessary for obtaining said protein and extraction of said expressed protein, analogues or derivatives from the culture medium of said transformed cell or from the cell extracts of said transformed cell. 5. Protein, or analogues and derivatives thereof, characterized by the ability to carry out the intracellular domain of the TNF or FAS receptor encoded by a DNA sequence according to claim 1 or produced according to the method in claim 4. 6. Use of one or more proteins, analogues or derivatives selected from the group consisting of the proteins, analogues and derivatives according to claim 5 and a protein which is p55IC, p55DD, FAS-IC or FAS-DD, analogues or derivatives thereof, all said proteins are capable of binding to the intracellular domain and modulating the activity of said TNF-R or FAS-R, for the preparation of a pharmaceutical composition for modulating the TNF or FAS-R ligand effect on cells bearing a TNF-R or a FAS-R, where said pharmaceutical composition comprises said one or more proteins, analogues or derivatives in a form suitable for intracellular introduction into cells, or a DNA sequence encoding said one or more proteins, analogues or derivatives in the form of a suitable vector carrying said sequence, said vector being able to cause insertion of said sequence into said cells in such a way that said sequence is expressed in said cells. 7. Use of an oligonucleotide sequence selected from a sequence that codes for an antisense sequence of at least part of the sequence according to claim 1, and a sequence that codes for the antisense sequence of p55IC, p55DD, FAS-IC or FAS-DD, said oligonucleotide sequence being in able to block the expression of at least one of the TNF-R or FAS-R intracellular domain binding proteins for the preparation of a pharmaceutical composition for modulating the TNF or FAS-R ligand action on cells bearing a TNF-R or a FAS-R. 8. Use of a recombinant animal virus vector carrying a sequence encoding a viral surface protein capable of binding to a specific tumor cell surface receptor or HIV-infected cell surface receptor or receptor carried by other diseased cells and a sequence encoding a protein, analog or derivative according to claim 5, which, when expressed in said tumor cell, HIV-infected cell or other diseased cell, is capable of killing said cell, for the preparation of a pharmaceutical composition for the treatment of tumor cells or HIV-infected cells or other diseased cells cells. 9. Use of one or more proteins, analogues or derivatives according to claim 5, for the production of a pharmaceutical composition for the induction of TNF-associated effects in cells or tissues, where said pharmaceutical composition comprises said one or more proteins, analogues or derivatives in a form suitable for intracellular introduction into cells, or a DNA sequence encoding said one or more proteins, analogues or derivatives in the form of a suitable vector carrying said sequence, said vector being able to cause insertion of said sequence into said cells in such a way that said sequence is expressed in said cells. 10. Use of a vector encoding a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding a protein according to claim 5 or an mRNA sequence encoding p55IC, p55DD, FAS-IC or FAS-DD for production of a pharmaceutical composition for modulating the TNF or FAS-R ligand effect on cells designed in a manner that allows expression of said ribozyme sequence in said cells, and when said ribozyme sequence is expressed in said cells it interacts with said cellular mRNA sequence and cuts said mRNA sequence, resulting in expression inhibition of said protein or said p55IC, p55DD, FAS-IC or FAS-DD in said cells. 11. Method for isolating and identifying proteins capable of binding the intracellular domain-binding proteins according to claim 5, comprising the use of the yeast two-hybrid procedure where a sequence coding for said intracellular domain-binding protein is carried by one hybrid vector and sequence from a cDNA or genomic DNA library is carried by the second hybrid vector, the vectors are then used to transform yeast host cells and the positively transformed cells are isolated, followed by extraction of said second hybrid vector to obtain a sequence encoding a protein that binds to said intracellular domain binding protein . 12. Pharmaceutical composition for modulating the TNF or FAS-R ligand effect on cells comprising a protein according to claim 5 or the protein p55IC, p55DD, FAS-IC or FAS-DD, its biologically active fragments, analogues, derivatives or mixtures thereof. 13. Pharmaceutical composition for modulating the TNF or FAS-R ligand action on cells comprising a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding a protein according to claim 5, or the protein p55IC, p55DD, FAS-IC or FAS-DD, its biologically active fragments or analogues. 14. Pharmaceutical composition for modulating the TNF or FAS-R ligand action on cells comprising an oligonucleotide sequence encoding an antisense sequence according to the sequence according to claim 1. 15. Pharmaceutical composition according to claim 12 for the treatment of cells by induction therein of TNF-associated effects, comprising p55-IC, parts thereof, analogues and derivatives thereof and a pharmaceutically acceptable carrier. 16. Use of a protein according to claim 5 for the preparation of a pharmaceutical composition for the treatment of tumors.