WO2002008270A2

WO2002008270A2 - A mort-1 interacting protein, its preparation and use

Info

Publication number: WO2002008270A2
Application number: PCT/IL2001/000649
Authority: WO
Inventors: David Wallach; Eugene Varfolomeev; Marcus Schuchmann
Original assignee: Yeda Research And Development Co. Ltd.
Priority date: 2000-07-26
Filing date: 2001-07-16
Publication date: 2002-01-31
Also published as: AU2001272732A1; WO2002008270A3; IL137524A0

Abstract

MORT-1 interacting proteins capable of modulating NF-λB activation and JNK activity are provided, together with their recombinant production.

Description

A MORT-1 INTERACTING PROTEIN, ITS PREPARATION AND USE

FIELD OF THE INVENTION

The present invention relates to a DNA sequence encoding a MORT-1 interacting protein capable of modulating NF-κB and JNK activities. More specifically, it relates to cDNA sequences encoding a biologically active protein herein designated EVPR (encoded by Clone 19), and isoforms, fragments, analogs and derivatives thereof capable of modulating NF-κB activation and JNK activity.

The invention also relates to the proteins encoded by the above DNAs, their preparation and the use of said proteins and DNA sequences in the treatment or prevention of conditions associated with NF-κB activation, c-Jun induction, or with any other activity mediated by NF-κB or by c-Jun, or with other molecules to which said protein binds. It also relates to antibodies to EVPR.

BACKGROUND OF THE INVENTION The Tumor Necrosis Factor/Nerve Growth Factor (TNF/NGF) receptor superfamily represents a growing family with over 20 members identified so far in mammalian cells. Although the receptors of this superfamily differ in the primary sequence of their extracellular domains, these domains share a conserved sequence motif (the cysteine rich subdomain) that is thought to adopt a conserved tertiary folds (Bazan, 1993; Beutler and van Huffel, 1994; Smith et al, 1994). In spite of their structural heterogeneity, there is much similarity of function between the receptors, indicating that they share common signaling pathways. One example for this similarity is the ability of several receptors of the TNF/NGF family to activate the transcription factor NF-κB through a shared set of signaling molecules.

While it is known that the tumor necrosis factor (TNF) receptors, and the structurally-related receptor Fas, trigger in cells, upon stimulation by leukocyte- produced ligands, destructive activities that lead to their own demise, the mechanisms of this triggering are still little understood. Mutational studies indicate that in the FAS- R and in the p55 TNF receptor (p55-R) signaling for cytotoxicity involves distinct regions within their intracellular domains (Brakebusch et al., 1992; Tartaglia et al, 1993; Itoh and Nagata, 1993). These regions (the 'death domains') have sequence similarity. The 'death domains' of both FAS-R and p55-R tend to self-associate. Their self-association apparently promotes that receptor aggregation which is necessary for initiation of signaling (see Song et al, 1994; Wallach et al, 1994; Boldin et al, 1995), and at high levels of receptor expression can result in triggering of ligand-independent signaling (Boldin et al, 1995).

Some of the cytotoxic effects of lymphocytes are mediated by interaction of the FAS-R ligand with Fas, a widely occurring cell surface receptor capable of triggering cell death (see also Nagata and Golstein, 1995). Cell killing by mononuclear phagocytes involves a ligand-receptor couple, TNF and its receptor p55-R (CD120), which are structurally related to FAS-R and its ligand (see also Vandenabeele et al., 1995). Three proteins that bind to the intracellular domain of FAS-R and p55-R at the region 'death domain' involved in cell-death induction by the receptors and that independently are also capable of triggering cell death were identified by the yeast two-hybrid screening procedure. One of these is MORT-1 (or FADD; WO 96/1864 and Boldin et al. 1995b), that binds specifically to Fas. The second one, TRADD (see also Hsu et al., 1995), binds to p55-R, and the third, RIP (see also Stanger et al, 1995), binds to both FAS-R and p55-R. Besides their binding to FAS-R and p55-R, these proteins are also capable of binding to each other, which provides for a possible functional "cross-talk" between FAS-R and p55-R. These bindings occur through a conserved sequence motif, the 'death domain module' common to the receptors and their associated proteins. Furthermore, although in the yeast two-hybrid test MORT-1 was shown to bind spontaneously to Fas, in mammalian cells, this binding takes place only after stimulation of the receptor, suggesting that MORT-1 participates in the initiating events of FAS-R signaling. MORT-1 does not contain any sequence motif characteristic of enzymatic activity, and therefore, its ability to trigger cell death does not seem to involve an intrinsic activity of MORT-1 itself, but rather, activation of some other protein(s) that bind MORT-1 and act further downstream in the signaling cascade.

Cellular expression of MORT-1 mutants lacking the N-terminal part of the molecule has been shown to block cytotoxicity induced by FAS-R or p55-R (Hsu et al., 1996; Chinnaiyan et al., 1996), indicating that this N-tenninal region transmits the signalling for the cytocidal effect of both receptors through protein-protein interactions.

Thus, the 'death domain' motifs of the receptors p55-R and FAS-R as well as their three associated proteins MORT-1, RIP and TRADD appear to be the sites of protein-protein interactions. The three proteins MORT-1, RIP and TRADD interact with the p55-R and FAS-R intracellular domains by the binding of their death domains to those of the receptors. Death domains of both RIP and TRADD can also self-associate while MORT-1 differs in that its death domain does not self-associate. Further, MORT- 1 and TRADD bind differentially to FAS-R and p55-R and also bind to each other. Moreover, both MORT-1 and TRADD bind effectively to RIP. Accordingly, it would seem that the interaction between the three proteins MORT-1, RIP and TRADD is an important part of the overall modulation of the intracellular signalling mediated by these proteins. Interference of the interaction between these three intracellular proteins will result in modulation of the effects caused by this interaction. For example, inhibition of TRADD binding to MORT-1 may modulate the Fas-p55-R interaction. Likewise, inhibition of RIP in addition to the above inhibition of TRADD binding to MORT-1 may further modulate Fas-p55-R interaction.

Using MORT-1 as bait in two hybrid screening the applicants identified a

MORT-1 binding protein (see WO 97/03998 and Boldin et al. 1996) which was first named MORT-2, and is now known as MACH/FLICE/Mch5 or Caspase 8.

MACH/Caspase 8 binds to MORT-1 and exists in multiple isoforms, some which contain a region that has proteolytic activity and shows marked sequence homology to proteases of the ICE/CED-3 family. Cellular expression of the proteolytic MACH isoforms results in cell death. Expression of MACH isoforms that contain an incomplete ICE/CED-3 region provides effective protection against the cytotoxicity induced by FAS-R or p55-R triggering. These findings suggest that MACH/Caspase 8 is the most upstream enzymatic component in the FAS-R and p55-R-induced cell death signalling cascades. Caspases are a family of evolutionarily conserved cysteine proteases that cleave proteins at specific substrate sites downstream of aspartate residues. Caspases play crucial roles in apoptotic processes and in the formation of several proinflammatory mediators (reviewed in Nicholson and Thornberry, 1997). Caspases are present in cells as inactive precursors, yet upon death induction become activated by processing at internal caspase substrate sites, allowing a cascade-like caspase activation process. The precursors of some of the caspases bind through the region upstream of their protease moiety to regulatory proteins that control their processing. Three caspases have been found to associate through motifs in these prodomains to homologous motifs found in adapter proteins of the p55-R (CD 120a) and CD95 signalling complexes. Caspase-8 (MACH/FLICE/Mch5) (Boldin et al. 1996, Muzio et al, 1996, Femandes- Alnemri et al, 1996) and caspase-10 (FLICE2/Mch4) (Fernandes-Alnemri et al, 1996, Vincenz and Dixit, 1997) bind through duplicated N-terminal death effector domains (DEDs) to an N-terminal DED in MORT1/FADD, and caspase-2 binds through an N- terminal motif named CARD (see hereinbelow) to a CARD domain in RAIDD (Duan and Dixit, 1997, Ahmad et al, 1997). Apparently, all three caspases participate in the induction of death, while CASH (see WO 98/399935) serves as a regulator (an inhibitor, or - according to other studies - a stimulator) of the death process. MACH however is currently the only protein for which direct evidence shows an involvement in death induction and recruitment to the FAS-R signalling complex (Muzio et al, 1996). Moreover, the present inventors have shown that targeted disruption of the MACH gene ablates death induction by TNF or by ligation of FAS-R or of DR3 (another death- inducing receptor of the TNF/NGF family, Varfolomeev et al, 1998).

The processing of MACH upon ligation of FAS-R or p55-R seems to result from juxtaposition of the MACH molecules recruited to the receptors, apparently through the mild proteolytic activity of the unprocessed MACH molecules themselves (Medema et al, 1997, Muzio et al, 1998). In vitro, MACH is capable of processing and activating almost all other caspases (Srinivasula et al, 1996). Within cells, however, it seems to act in a much more restricted manner, resulting in the sequential activation, first of caspase-9 (Pan et al, 1998), then of caspase-3 and caspase-7, and later of caspase-6 (Hirata et α/., 1998).

Adapter molecules such as the serine/threonine (Ser/Thr) kinase RIP mediate divergent signalling pathways such as for NF-kB activation and for cell death through specific interactions with death domain containing adapter proteins and adapter proteins of the tumor necrosis receptor family - associated factors (TRAFs). A 61-kDa protein kinase related to RIP that seems to be a component of both the TNFR-1 and the CD40 signalling complexes was recently identified. Receptor interacting protein-2 (RIP2), also known as Bl/ CARDIAKJRICK (see WO 98/55507, Inohara et al 1998, Thome et al 1998) contains an N-terminal domain with homology to Ser/Thr kinases and a C- terminal caspase activation and recruitment domain (CARD), a homophilic interaction motif that mediates the recruitment of caspase death proteases. Overexpression of RIP2 signalled both NF-κB activation and cell death. Mutational analysis revealed the pro- apoptotic function of RIP2 to be restricted to its C-terminal CARD domain, whereas the intact molecule was necessary for NF-κB activation. RIP2 interacted with other members of the TNFR-1 signalling complex, including inhibitor of apoptosis protein cIAPl and with members of the TNFR-associated factor (TRAF) family, specifically TRAF1, TRAF5, and TRAF6, but not with TRAF2, TRAF3, or TRAF4. These TRAF interactions apparently mediate the recruitment of RIP2 to receptor signalling complexes. It has also been found that besides the above noted cell cytotoxicity activities and modulation thereof mediated by the various receptors and their binding proteins including Fas, p55-R, MORT-1, TRADD, RIP, MACH, Mch4, a number of these receptors and their binding proteins are also involved in the modulation of the activity of nuclear transcription factor NF-κB. NF-κB is a key mediator of cell survival or viability, being responsible for the control of expression of many immune- and inflammatory- response genes. TNF-α can stimulate activation of NF-κB and thus TNF- α is capable of inducing two kinds of signals in cells, one eliciting cell death and another that protects cells against death induction by inducing gene expression via NF- KB (see Beg and Baltimore, 1996; Wang et al., 1996; Van Antwerp et al, 1996). A similar dual effect for FAS-R has also been reported (see reference to this effect as stated in above Van Antwerp et al., 1996). It would therefore appear that there exists a delicate balance between cell death and cell survival upon stimulation of various types of cells with TNF-α and/or the FAS-R ligand, the ultimate outcome of the stimulation depending on which intracellular pathway is stimulated to a greater extent, the one leading to cell death (usually by apoptosis), or the one leading to cell survival via activation of NF-κB.

In addition, the applicants have also recently further elucidated the possible pathway by which members of the TNF/NGF receptor family activate NF-κB (see Malinin et al., 1997 and the various relevant references set forth therein; and co- owned, co-pending Israel Patent Application Nos. IL 117800 and IL 119133). As described there, it arises that several members of the TNF/NGF receptor family are capable of activating NF-κB through a common adaptor protein, TRAF2. A newly elucidated protein kinase called NIK (see above Malinin et al., 1997 and WO 97/37016) is capable of binding to TRAF2 and of stimulating NF- B activity. In fact, it was shown (see aforesaid Malinin et al. and IL applications) that expression in cells of kinase- deficient NIK mutants results in the cells being incapable of having stimulation of NF- KB in a normal endogenous manner and also in the cell having a block in induction of NF-κB activity by TNF, via either FAS-R, and a block in NF- B induction by TRADD, RIP and MORT-1 (which are adaptor proteins that bind these p55-R and/or Fas receptors see above). All of the receptors p55-R, ρ75-R, FAS-R and their adaptor proteins MORT-1, TRADD and RIP bind directly or indirectly to TRAF2, whose binding to NIK may contribute to the induction of NF-κB.

NIK was shown to share sequence similarity with MAP3K kinases and was suggested to participate in the NF-κB inducing signalling cascade common to receptors of the TNF/NGF family and to the IL-1 type 1 receptor. TNF-α and IL-lβ, initiate a signalling cascade leading to activation of two IκB kinases, IKK-1 (IKK-α) and IKK-2 (IKK-β), which phosphorylate IκB at specific N-terminal serine residues (S32 and S36 for IκBα S19 and S23 for IκBβ, for review see Mercurio F and Manning AM, 1999). These kinases were identified as the components of a high molecular weight protein complex designated the IKK signalsome.

Kinases of the mitogen-activated protein kinase (MAPK) family have been shown to be key players for signalling pathways that are triggered by TRAF-containing complexes. One of the targets of these pathways is c-Jun amino-(N)-terminal kinase (JNK) activation (Reinhard etal. 1997; Song et al. 1997). TRAF proteins can thus serve to modulate the ability of receptors to trigger distinct signalling pathways that lead to phosphorylation and activation of protein kinases and, subsequently, to the activation of transcription factors of the Rel and AP-1 family.

The c-Jun transcription factor is phosphorylated at its amino terminus by JNK, the most downstream member of one MAPK signalling pathway (Hibi et al. 1993). To be activated, JNK needs to be phosphorylated by a MAPK kinase (MAPKK, SEK, MEK). This kinase itself is phosphorylated by a MAPKKK (MAP3K MEKK1). The mode of activation of the MAP3Ks is yet to be clarified. In several systems, activation involves small G proteins. These proteins apparently prompt phosphorylation of the MAP3Ks by other, heterogeneous kinases (MAP4Ks). The substrates affected by these cascades are highly heterogeneous, some themselves being protein kinases (dubbed MAPKAPKs). Mammalian cells contain three major known MAPK cascades (reviewed in Ichijo 1999). The extracellular signal regulated kinase (ERKs), the c-Jun amino terminal kinases (JNKs, also referred to as stress-activated protein kinases or SAPKs) and the p38 MAPKs (p38s). All are activated by both the TNF and the FAS-R systems, though the components of the activated cascades, as well as their targets, may vary from one cell type to another, in keeping with the cell type-specific patterns of responses to the TNF/Fas systems. The three cascades have different functions but cross-react on several levels. Although many JNK/p38-activating stimuli are proapoptotic, the biological outcome of JNK/p38 is highly divergent and appears to be largely dependent on the cell type or cellular context. As implied by their name, the SAPK cascades induce adaptive responses to a variety of stress signals mainly by inducing changes in gene expression. The SAPKl/JNK pathway participates in the regulation of gene expression by the TNF/Fas systems both by enhancing the function of transcription factors, of which the most thoroughly studied is API (Karin, 1996), and by affecting the stability of certain RNA transcripts (Chen et al, 1998). It affects transcription through the phosphorylation of various transcription factors, including c-Jun, ATF2, Elk-1, and CREB and affects genes such as collagenase IL-1 and c-Jun.

As prolonged JNK activation may result in death of some cells it was suggested that this enzyme might be involved in the TNF/Fas-induced signalling for death. Similar to various other activities of the TNF and Fas systems, their activating effect on the

SAPK cascades is regulated by antagonising mechanisms and is therefore mostly transient, followed by a rather long period of inactivity. TNF-induced activation of phosphatase(s) could contribute to the transient character of SAPKl/JNK activation (Guo et al, 1998). Many of the receptors of the TNF/NGF family can activate the

SAPKl/JNK cascade (e.g. see Marsters et al, 1997, Reinhard et al, 1997). The effect of the TNF receptors involves TRAF2 (Yeh et al, 1997), and according to limited evidence also MADD (Schievella et al, 1997). The effect of CD95 involves the DD- associated adaptor protein DAXX (Yang et al, 1997, Chang et al, 1998). In addition, the SAPKl/JNK pathway can be activated late in the process of death induction through caspase-mediated processing and activation of kinases that act in this pathway, for example PAK2 (Rudel and Bokoch, 1997), PAK65 (Lee et al, 1997) and MEKK1

(Cardone et al, 1997, Deak et al, 1998).

In all in vivo studies reported so far, induction of the p38 and JNK pathways has occurred simultaneously. In certain in vivo situations, however, it is possible to observe differential responses of the two pathways to some stimuli, suggesting that they share both common and distinct activation mechanisms.

The MAP2Ks activating the p38 kinases were reported to be MKK2 and MKK3

(in response to TNF; Winston et al, 1991) and MKK6 (in response to CD95 ligation, Toyoshima et al, 1997). As in the case of the JNK pathway, these kinases are activated by the TNF/Fas systems in both a caspase-dependent and a caspase-independent manner (Toyoshima et al, 1997, Juo et al, 1997). Of the two known caspase-independent JNK- activation pathways, the one involving GCK (or GCKR) and MEKK1 seems to lead rather specifically to JNK activation (see Yuasa et al. 1998 and references within). The other, mediated by ASKl, can activate p38 as well (Carpentier et al, 1998). Occurrence of a signalling pathway that can mediate specific activation of p38 by TNF was suggested in a recent report describing association of a "p38 specific" MAP3K activity with the intermediate region of RIP (the region linking its death domain to the kinase domain in RIP). The identity of the RIP-associated enzyme mediating this p38-specifιc function is still unknown (Yuasa et al, 1998).

Dominant-negative mutants of either of these proteins that lack kinase activity block TRAF -mediated JNK activation that is induced by members of the TNF/NGFR superfamily. Thus, TRAF proteins appear to regulate the JNK activation pathway at a very proximal step (Liu et al. 1996; Lee et al. 1997; Reinhard et al. 1997). Cells from TRAF2-deficient mice failed to activate JNK in response to TNFα (Yeh et al. 1997).

SUMMARY OF THE INVENTION

It is an object of the invention to provide a novel protein EVPR (encoded by Clone 19), including isoforms, analogs, fragments or derivatives thereof, capable of binding to the MORT-1 protein (herein after 'MORT-1'). As MORT-1 is capable of interacting directly or indirectly with the intracellular mediators of inflammation, cell cytotoxicity/cell death, such as p55-R and FAS-R and their associated adaptor or modulator proteins such as, for example, TRADD, MACH, Mch4 and others, the novel proteins of the present invention by binding to MORT-1 are therefore capable of affecting the intracellular signaling process initiated by the binding of ligands of the TNF family to death domain containing receptors (such as Fas or the p55-R), and as such the new proteins of the present invention are modulators of the cellular capability to interact with MORT-1. Despite their effects of these receptors, the new proteins do not possess a death domain. The present invention thus provides a DNA sequence encoding a MORT-1 interacting protein, herein designated EVPR, or isoforms, fragments, analogs or derivatives thereof, said protein capable of modulating NF-κB activation and of JNK activity, and lacking a death domain. Preferably, the DNA sequence is selected from the group a) a cDNA sequence of the herein designated murine clone 19a (depicted in fig. 1), b) a cDNA sequence of the herein designated murine clone 19c (depicted in fig- 1), c) a cDNA sequence of the herein designated murine clone 19d (depicted in fig- 1), d) a cDNA sequence of the herein designated human clone 19a (depicted in fig- 1), e) a cDNA sequence of the herein designated human clone 19b (depicted in fig. 1), f) a fragment of sequence (a)-(e), g) a DNA sequence capable of hybridization to a sequence of (a)-(f) under stringent conditions, h) a DNA sequence which is degenerate as a result of the genetic code to the DNA sequences defined in (a)-(g).

In particular, the DNA sequence may comprise nucleotides 111 to 2493 of human clone 19b depicted in fig. 1.

The invention also provides a vector comprising a DNA sequence as above, capable of being expressed in a eukaryotic or prokaryotic host cell.

Such transformed eukaryotic or prokaryotic host cells are also provided by the present invention. The invention further provides a MORT-1 interacting protein, herein designated EVPR, isoforms, fragments, analogs or derivatives thereof, capable of modulating NF- KB activation and of JNK activity, said protein lacking a death domain.

In particular, the protein according to the invention is encoded by any of the above DNA sequences.

Preferably, the protein according to the invention is encoded by at least nucleotides 111 to 2493 of human clone 19b depicted in fig. 1.

The invention also provides a method for producing a protein, isoform, fragment, analog or derivative thereof, capable of interacting with MORT-1 and of modulating NF-κB and JNK activity, comprising growing a transformed host under conditions suitable for expression of said protein, isoform, fragment, analog or derivative thereof, effecting post-translational modifications, as necessary, for obtaining said protein, isoform, fragment, analog or derivative thereof, and isolating said protein, isoform, fragment, analog or derivative thereof. In an embodiment of the present invention there are provided antibodies specific for a MORT-1 interacting protein, herein designated EVPR, isoforms, fragments, analogs or derivatives thereof, capable of modulating NF-κB activation and of JNK activity, said protein lacking a death domain.

Furthermore, the invention provides a method for the modulation in cells of the activity of NF-κB or JNK, said method comprising treating said cells by introducing into said cells one or more of a protein, according to the invention, isoform, analog, fragment or derivative thereof, in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more protein, isoform, analog, fragment or derivative thereof in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.

In accordance with the invention such a method may comprise introducing into said cells a DNA sequence encoding said protein, isoform, fragment, analog or derivative in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.

In particular, the method may comprise the steps of :

(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of said cells to be treated and a second sequence encoding a protein selected from the said protein, isoforms, analogs, fragments and derivatives according to any one of claims 8-10, that when expressed in said cells is capable of modulating/mediating the activity of NF-κB or any other intracellular signaling activity modulated/mediated by TRAF2 or other said molecules; and

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

The invention also provides a pharmaceutical composition for the modulation of the activity of NF-κB or JNK comprising, as active ingredient at least one MORT-1 interacting protein, herein designated EVPR, isoforms, fragments, analogs or derivatives thereof, capable of modulating NF-κB activation and of JNK activity, said protein lacking a death domain.

The pharmaceutical composition may also comprise, as active ingredient, a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding at least one MORT-1 interacting protein, herein designated

EVPR, isoforms, fragments, analogs or derivatives thereof, capable of modulating NF-

KB activation and of JNK activity, said protein lacking a death domain.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the nucleotide sequence of murine clone 19 variants a, c and d and human clone 19 variants a and b. Murine clone 19or extends from nucleotides 1-517 (as marked), while clone 19ex corresponds to murine clone 19a. Figure 2 shows the deduced amino acid sequence alignment of murine and human clone 19 variants, with the ESTs found in the databases.

Figure 3 shows the enhancement of TRADD and p55TNFR NF-κB induction by clone 19 measured by arbitrary units of luciferase activity in four reporter gene assay experiments. Figure 4 shows the prolongation of JNK activation (in Hela cells) and enhancement of JNK activity (in 293 T cells) by clone 19ex upon treatment of transfected cells with TNF (10000 U/ml).

Figure 5 top panel shows the prolongation of JNK activation (in Hela cells) by clone 19ex upon treatment of transfected cells with TNF (10000 U/ml). TNF does not potentiate clone 19or induced JNK activation. Lower panel shows that TRAF -2 dominant negative (DN) mutant downregulates clone 19or and clone 19ex TNF induced JNK activation in Hela Bujard cells

Figure 6 shows the inhibition of Bl (RIP 2) kinase induction of NF-κB by clone 19 measured by arbitrary units of luciferase activity in three independent reporter gene assay experiments.

Figure 7 shows Clone 19or and Clone 19ex inhibition of RIP-2-induced JNK activation.

Figure 8 shows the mRNA expression of clone 19 as detected in northern blot analysis of adult and embryonic mouse tissues. The arrow to the right shows the major clone 19 transcript. DETAILED DESCRIPTION OF THE INVENTION

To isolate proteins interacting with MORT-1, the yeast two-hybrid system was used (Fields and Song, 1989). The two-hybrid system is a yeast-based genetic assay for the detection of specific protein-protein interactions in vivo by restoration of an eukaryotic transcriptional activator such as GAL4 that has two separate domains, a DNA binding and an activation domain. The above-noted domains, when expressed and bound together, form a restored GAL4 protein, which is capable of binding to an upstream activating sequence which in turn activates a promoter that controls the expression of a reporter gene, such as lacZ or HIS3, the expression of which is readily observed in the cultured cells. In this system, the genes for the candidate interacting proteins are cloned into separate expression vectors. In one expression vector, the sequence of one candidate protein is cloned in frame with the sequence of the GAL4 DNA-binding domain to generate a hybrid protein with the GAL4 DNA-binding domain, and in the other vector, the sequence of the second candidate protein is cloned in frame with the sequence of the GAL4 activation domain to generate a hybrid protein with the GAL4-activation domain. The two hybrid vectors are then co-transformed into a yeast host strain having a lacZ or HIS3 reporter gene under the control of upstream GAL4 binding sites. Only those transformed host cells (co-transformants) in which the two hybrid proteins are expressed and are capable of interacting with each other, will be capable of expressing the reporter gene. In the case of the lacZ reporter gene, host cells expressing this gene will become blue in color when X-gal is added to the cultures. Hence, blue colonies are indicative of the fact that the two cloned candidate proteins are capable of interacting with each other.

Using this two-hybrid system, MORT-1, was cloned into the vector pGBT9 (carrying the GAL4 DNA-binding sequence, provided by Clontech, USA, see below), to create fusion proteins with the GAL4 DNA-binding domain. For the cloning of MORT- 1 into pGBT9, a clone encoding the full-length cD A sequence of human MORT1 (accession number X84709) was inserted into the pGBT9 vector opened in its multiple cloning site region (MCS), with the corresponding suitable restriction enzymes. The above hybrid (chimeric) vector was then cotransfected together with an oligodT and random primed cDNA library prepared from 11.5 days old mouse embryos cloned into the pGAD GH vector bearing the GAL4 activating domain, into a HF7c yeast host strain. All the above-noted vectors, pGBT9 and pGAD GH carrying the mouse embryo cDNA library, and the yeast strain were purchased from Clontech Laboratories, Inc., USA, as a part of MATCHMAKER Two-Hybrid System, #K1605-1. The co-transfected yeast were selected for their ability to grow in medium lacking Histidine (His^" medium), growing colonies being indicative of positive transformants. The selected yeast clones were then tested for their ability to express the lacZ gene, i.e., for their LACZ activity, by adding X-gal to the culture medium, which is catabolized to form a blue colored product by β-galactosidase, 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 transcription activator be present in an active form in the transformed clones, i.e. that the GAL4 DNA-binding domain encoded by the above hybrid vector be combined properly with the GAL4 activation domain encoded by the other hybrid vector. Such a combination is only possible if the two proteins fused to each of the GAL4 domains are capable of stably interacting (binding) with each other. Thus, the His and blue (LacZ ) colonies that were isolated are colonies which have been cotransfected with a vector encoding MORT-1 and a vector encoding a protein product of murine embryo origin that is capable of stably binding to MORT-1.

The plasmid DNA from the above His , LACZ yeast colonies was isolated and electroporated into E. coli strain HB101 by standard procedures followed by selection of Leu and Ampicillin resistant transformants, these transformants being the ones carrying the hybrid pGAD GH vector which has both the Amp^R and Leu2 coding sequences. Such transformants therefore are clones carrying the sequences encoding newly identified proteins capable of binding to MORT-1. Plasmid DNA was then isolated from these transformed E. coli and re-tested by:

(a) Re-transforming them with the original MORT-1 hybrid plasmid (hybrid pGTB9 carrying MORT-1) into yeast strain HF7 as set forth hereinabove. As controls, vectors carrying irrelevant protein encoding sequences, e.g., pACT-lamin or pGBT9 alone were used for cotransformation with the found MORT-1 -binding protein- encoding plasmid which was designated Clone 19. The cotransformed yeasts were then tested for growth on His^" medium alone, or with different levels of 3-aminotriazole; and (b) re-transforming the plasmid DNA and original MORT-1 hybrid plasmid and control plasmids described in (a) above into yeast host cells of strain SFY526 and determining the LACZ activity (efficiency of β-gal formation, i.e., blue color formation). The results of the above tests revealed that the pattern of growth of colonies in His^" medium was identical to the pattern of LacZ activity, as assessed by the color of the colony, i.e., His⁺ colonies were also LacZ⁺. Further, the LacZ activity in liquid culture (preferred culture conditions) was assessed after transfection of the GAL4 DNA-binding and activation-domain hybrids into the SFY526 yeast hosts which have a better LACZ inducibility with the GAL4 transcription activator than that of the HF7 yeast host cells.

The original cDNA of clone 19 (clone 19or for original, herein) was cloned by the abovementioned two hybrid screening method. The so-isolated clone 19-cDNA insert was then sequenced using standard DNA sequencing procedures and found to contain 517 nucleotides encoding a protein with a deduced 170 amino acids sequence. The amino acid sequence of clone 19 was deduced from the DNA sequence using DNA Strider V1.2 software.

An additional clone 19 cDNA was cloned by PCR from the above noted library by using appropriate PCR-primers, designed by OLIGO5™ software. A sense primer encoding a sequence from the 3' end sequence of the original clone 19 and an antisense primer derived from the pGAD GH vector sequences were used. The cDNA clone obtained was found to contain an additional 260bp at the 3' end of the sequence of clone 19or. Altogether the two cDNA clones encode a protein with a deduced sequence of about 260 amino acids. A cDNA clone comprising nucleotides 1 to 777, which encode the 260 amino acids was constructed, and is herein-designated clone 19ex, for extended.

In order to evaluate the interaction of clone 19 with other proteins that mediate the

TNF-induced signalling cascade a two-hybrid test in yeast was performed. After growing the bacteria and extracting and purifying the appropriate plasmid DNA therefrom, the plasmid DNA was then transformed into SFY526 yeast cells. The lacZ activity of SFY526 transformants was then tested by plating on selection medium including histidine and containing Xgal. The yeast colonies were then lifted using a Whatman 3MM No. 50 filter paper (for a description of colony lifting, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989), then placed for about 20 sec on aluminium foil, transferred for about 25 sec to liquid nitrogen in order to freeze the yeast Cells, exposed for about 1 min. to room temperature in order to thaw the yeast cells, placed in a petri dish on a Whatmann 3MM No. 1 filter paper which was previously soaked in Z buffer (beta- galactosidase reaction buffer, see e.g., Clontech protocols, the above Sambrook et al., or the Ausubel et al, Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, NY, 2000) containing Xgal and beta-mercaptoethanol. The appearance of blue colour in the colonies is an indication of active beta- galactosidase. Clone 19 interacting proteins usually develop blue colour in this assay within 1 hour to overnight. The two-hybrid test in yeast was performed by cloning clone 19or and clone 19ex into the GAD vector used for cloning the library (the "prey" vector) or into the GBT vector used for expressing the "bait" used for screening of the library. The two-hybrid tests in yeast revealed that clone 19or and clone 19ex strongly interact with themselves, with the full length MORT1 as well as with the full length and Death Domains (DD) of RIP, TRADD, p55-R, and Fas. LPR-like mutations which disrupt the structure of the FAS-R death domain (Varfolomeev et al, J. Exp. Med., 183, 1271-1275, 1995) were introduced into the DD of RIP, p55-R, and Fas. These mutations abrogated binding of the mutant proteins with clone 19 in the two-hybrid test.

Functional characterisation of clone 19 involved analyzing its cytotoxic inducing effects as well as its effects on modulation of cytotoxicity induced by genes that mediate the TNF-induced signalling cascade. Transient cotransfection of HEK 293 -T cells with clone 19or (and clone 19ex) did not induce cytotoxicity, nor did it affect cytotoxicity induced by co-expression of the p55TNFR, Fas, TRADD, MORT, RIP, and MACH in HEK-293T cells.

Cotransfection of clone 19 with known inducers of NF-κB activation. (TRADD and p55TNFR) resulted in some enhancement of NF- B induction. Cotransfection of clone 19 with RIP-2 (Bl kinase) led to a two to three fold reduction in NF-κB induction. Clone 19 also potentiated NF-κB induction by TRADD by 10-25 %.

It is acknowledged that the trans-activation potential of c-Jun is regulated by extracellular signal-induced phosphorylation of two serine residues (⁶³Ser & ⁷³Ser) of its amino-terminal activation domain. The JNK/SAPK protein kinases responsible for the abovementioned phosphorylation constitute a subset of the MAP kinase family and are themselves activated via phosphorylation at ¹⁸³Thr and ¹⁸ Tyr mediated by further upstream dual-specificity kinases. Therefore, the phosphorylation status of the appropriate sites within both c-Jun and JNKs can be used as a marker reflecting the activation state of the protein.

In order to examine the effect of clone 19 on TNF induced JNK activation, clone 19 was transiently co-transfected in TNF treated 293T cells and Hela-FAS-R cells, which constitutively express Fas, together with a HA- JNK 1 -expressing plasmid. It was found that upon treatment of transfected cells with TNF (lOOOOU/mt) clone 19 could prolong the duration of JNK activation in Hela-FAS-R cells and enhance JNK activity in 293T cells.

Clone 19, having the abovementioned effects on NF-κB and JNK activities can be used for modulating, i.e. enhancing or inhibiting JNK and NF-κB activities induced by TNF and or Fas ligand in vivo or in vitro. The present invention concerns the DNA sequence encoding a Clone 19 protein

(EVPR) and the EVPR proteins encoded by the DNA sequences.

Accordingly, a sequence encoding for an EVPR was cloned, by the use of PCR and chemical oligonucleotide synthesis. PCR allows for the amplification of specific DNA sequences by repeated DNA polymerase reactions (as in above Current Protocols in Molecular Biology). In order to carry out the PCR, sense primers were synthesized by automated DNA synthesis which were complementary to the sequence of the Clone 19 ex and antisense primers were synthesized which were complementary to the sequence of the murine EST clone homologous to the 3' end of the human clone 19. These primers were used in the PCR reaction using as template cDNA generated by reverse transcription from RNA extracted from mouse spleen. A short splice variant of murine clone 19 of approximately 1.5 Kb was thus cloned and sequenced.

Moreover, the present invention further concerns the DNA sequences encoding biologically active analogs, (such as muteins) fragments and derivatives of EVPR, and the analogs, fragments and derivatives encoded thereby. The preparation of such analogs, fragments and derivatives is by standard procedure (see for example, Sambrook et al., 1989) in which in the DNA sequences encoding EVPR, one or more codons may be deleted, added or substituted by another, to yield analogs having at least one amino acid residue change with respect to the native protein. It is understood that such analogs fragments or derivatives have the same biological activities as EVPR.

Of the above DNA sequences of the invention which encode EVPR, isoform, analog, fragment or derivative thereof, there are also included, as an embodiment of the invention, DNA sequences capable of hybridizing with a cDNA sequence derived from the coding region of a native EVPR, in which such hybridization is performed under stringent conditions, and which hybridizable DNA sequences encode a biologically active EVPR. These hybridizable DNA sequences therefore include DNA sequences which have a relatively high homology to a native Clone 19 cDNA sequence and as such represent Clone 19-like sequences which may be, for example, naturally-derived sequences encoding the various Clone 19 isoforms, or naturally-occurring sequences encoding proteins belonging to a group of Clone 19-like sequences encoding a protein having the activity of EVPR. Further, these sequences may also, for example, include non-naturally occurring, synthetically produced sequences, that are similar to a native Clone 19 cDNA sequence but incorporate a number of desired modifications. Such synthetic sequences therefore include all of the possible sequences encoding analogs, fragments and derivatives of EVPR, all of which have the activity of Clone 19.

Analogs or muteins in accordance with the present invention include proteins encoded by a nucleic acid, such as DNA or RNA, which hybridizes to DNA or RNA, which encodes an EVPR, in accordance with the present invention, under stringent conditions. The invention also includes such nucleic acid, which is also useful as a probe in identification and purification of the desired nucleic acid. Furthermore, such nucleic acid would be a prime candidate to determine whether it encodes a polypeptide, which retains the functional activity of an EVPR of the present invention. The term "stringent conditions" refers to hybridization and subsequent washing conditions, which those of ordinary skill in the art conventionally refer to as "stringent". See Ausubel et al, Current Protocols in Molecular Biolofiy, supra, Interscience, N.Y., §§6.3 and 6.4 (1987, 1992), and Sambrook et al., supra. Without limitation, examples of stringent conditions include washing conditions 12-20°C below the calculated Tm of the hybrid under study in, e.g., 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% SDS for 15 minutes; 0.1 x SSC and 0.5% SDS at 37°C for 30-60 minutes and then, a 0.1 x SSC and 0.5% SDS at 68°C for 30-60 minutes. Those of ordinary skill in this art understand that stringency conditions also depend on the length of the DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferable to use tetramethyl ammonium chloride (TMAC) instead of SSC. See Ausubel, supra.

To obtain the various above noted naturally occurring Clone 19-like sequences, standard procedures of screening and isolation of naturally-derived DNA or RNA samples from various tissues may be employed using the natural Clone 19 cDNA or portion thereof as probe (see for example standard procedures set forth in Sambrook et al., 1989). Likewise, to prepare the above noted various synthetic Clone 19-like sequences encoding analogs, fragments or derivatives of EVPR, a number of standard procedures may be used as are detailed herein below concerning the preparation of such analogs, fragments and derivatives.

A polypeptide or protein "substantially corresponding" to EVPR includes not only EVPR but also polypeptides or proteins that are analogs of EVPR.

Analogs that substantially correspond to EVPR are those polypeptides in which one or more amino acid of EVPR 's amino acid sequence has been replaced with another amino acid, deleted and/or inserted, provided that the resulting protein exhibits substantially the same or higher biological activity as EVPR to which it corresponds. In order to substantially correspond to EVPR, the changes in the sequence of EVPRs, such as isoforms are generally relatively minor. Although the number of changes may be more than ten, preferably there are no more than ten changes, more preferably no more than five, and most preferably no more than three such changes. While any technique can be used to find potentially biologically active proteins that substantially correspond to EVPRs, one such technique is the use of conventional mutagenesis techniques on the DNA encoding the protein, resulting in a few modifications. The proteins expressed by such clones can then be screened for their ability to modulate JNK and or NF-KB activity in modulation/mediation of the intracellular pathways noted above.

"Conservative" changes are those changes which would not be expected to change the activity of the protein and are usually the first to be screened as these would not be expected to substantially change the size, charge or configuration of the protein and thus would not be expected to change the biological properties thereof. Conservative substitutions of EVPRs include an analog wherein at least one amino acid residue in the polypeptide has been conservatively replaced by a different amino acid. Such substitutions preferably are made in accordance with the following list as presented in Table IA, which substitutions may be determined by routine experimentation to provide modified structural and functional properties of a synthesized polypeptide molecule while maintaining the biological activity characteristic of EVPR.

Table IA

Original Exemplary

Residue Substitution

Ala Gly;Ser

Arg Lys

Asn Gln;His

Asp Glu

Cys Ser

10 Gin Asn

Glu Asp

Gly Ala;Pro

His Asn;Gln

He Leu;Val

15 Leu Ile;Val

Lys Arg;Gln;Glu

Met Leu;Tyr;Ile

Phe Met;Leu;Tyr

20 Thr Ser

Trp Tyr

Tyr Trp;Phe

Val Ile;Leu Alternatively, another group of substitutions of EVPR are those in which at least one amino acid residue in the polypeptide has been removed and a different residue inserted in its place according to the following Table IB. The types of substitutions which may be made in the polypeptide may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1-2 of Schulz et al., G.E., Principles of Protein Structure Springer- Verlag, New York, NY, 1798, and Figs. 3-9 of Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, CA 1983. Based on such an analysis, alternative conservative substitutions are defined herein as exchanges within one of the following five groups:

TABLE IB

Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly);

2. Polar negatively charged residues and their amides: Asp, Asn, Glu, Gin;

3. Polar, positively charged residues: His, Arg, Lys;

4. Large aliphatic nonpolar residues: Met, Leu, He, Val (Cys); and

5. Large aromatic residues: Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles in protein architecture. Gly is the only residue lacking any side chain and thus imparts flexibility to the chain. This however tends to promote the formation of secondary structure other than a-helical. Pro, because of its unusual geometry, tightly constrains the chain and generally tends to promote β-turn-like structures, although in some cases Cys can be capable of participating in disulfide bond formation which is important in protein folding. Note that Schulz et al., supra, would merge Groups 1 and 2, above. Note also that Tyr, because of its hydrogen bonding potential, has significant kinship with Ser, and Thr, etc. Conservative amino acid substitutions according to the present invention, e.g., as presented above, are known in the art and would be expected to maintain biological and structural properties of the polypeptide after amino acid substitution. Most deletions and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the protein or polypeptide molecule. "Characteristics" is defined in a non-inclusive manner to define both changes in secondary structure, e.g. a-helix or β-sheet, as well as changes in biological activity, e.g., modulation and/or mediation of JNK and/or NF-κB activity.

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

Besides conservative substitutions discussed above which would not significantly change the activity of EVPR, either conservative substitutions or less conservative and more random changes, which lead to an increase in biological activity of the analogs of EVPRs, are intended to be within the scope of the invention.

When the exact effect of the substitution or deletion is to be confirmed, one skilled in the art will appreciate that the effect of the substitution(s), deletion(s), etc., will be evaluated by routine binding and cell death assays. Screening using such a standard test does not involve undue experimentation.

Acceptable Clone 19 analogs are those which retain at least the modulation and/or mediation of JNK and/or NF-κB activity, and thereby, as noted above mediate the activity of JNK and/or NF-κB activity in the intracellular pathways as noted above. In such a way, analogs can be produced which have a so-called dominant-negative effect, namely, an analog which is defective either in modulation and/or mediation of JNK and/or NF-i B, or in subsequent signalling or other activity following such modulation. Such analogs can be used, for example, to inhibit the effect of JNK, or to inhibit the NF-κB inducing or reducing (direct or indirect) effect of Clone 19, depending on which of these activities is the major one modulated by Clone 19 (see above), and this by such analogs competing with the activity Clone 19.

At the genetic level, these analogs are generally prepared by site-directed mutagenesis of nucleotides in the DNA encoding EVPR, thereby producing DNA encoding the analog, and thereafter synthesizing the DNA and expressing the polypeptide in recombinant cell culture. The analogs typically exhibit the same or increased qualitative biological activity as the naturally occurring protein, Ausubel et al., Current Protocols in Molecular Biology, Greene Publications and Wiley Interscience, New York, NY, 2000; Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

Preparation of an EVPR in accordance herewith, or an alternative nucleotide sequence encoding the same polypeptide but differing from the natural sequence due to changes resulting from the known degeneracy of the genetic code, can be achieved by site-specific mutagenesis of DNA that encodes an earlier prepared analog or a native version of an EVPR. Site-specific mutagenesis allows the production of analogs tlirough the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 20 to 25 nucleotides in length is preferred, with about 5 to 10 complementing nucleotides on each side of the sequence being altered. In general, the technique of site-specific mutagenesis is well known in the art, as exemplified by publications such as Adelman et al, DNA 2:183 (1983), the disclosure of which is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage, for example, as disclosed by Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), the disclosure of which is incorporated herein by reference. These phages are readily available commercially and their use is generally well known to those skilled in the art. Alternatively, plasmid vectors that contain a single-stranded phage origin of replication (Veira et al., Meth. Enzymol. 153:3, 1987) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the relevant polypeptide. An oligonucleotide primer bearing the desired mutated sequence is prepared synthetically by automated DNA/oligonucleotide synthesis. This primer is then annealed with the single-stranded protein-sequence- containing vector, and subjected to DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment, to complete the synthesis of the mutation-bearing strand. Thus, a mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells, such as E. coli JM101 cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement. After such a clone is selected, the mutated EVPR sequence may be removed and placed in an appropriate vector, generally a transfer or expression vector of the type that may be employed for transfection of an appropriate host.

Accordingly, gene or nucleic acid encoding for a EVPR can also be detected, obtained and/or modified, in vitro, in situ and/or in vivo, by the use of known DNA or RNA amplification techniques, such as PCR and chemical oligonucleotide synthesis. PCR allows for the amplification (increase in number) of specific DNA sequences by repeated DNA polymerase reactions. This reaction can be used as a replacement for cloning; all that is required is the knowledge of a nucleic acid sequence. In order to carry out PCR, primers are designed which are complementary to the sequence of interest. The primers are then generated by automated DNA synthesis. Because primers can be designed to hybridize to any part of the gene, conditions can be created such that mismatches in complementary base pairing can be tolerated. Amplification of these mismatched regions can lead to the synthesis of a mutagenized product resulting in the generation of a peptide with new properties (i.e., site directed mutagenesis). See also, e.g., Ausubel, supra, Ch. 16. Also, by coupling complementary DNA (cDNA) synthesis, using reverse transcriptase, with PCR, RNA can be used as the starting material for the synthesis of the extracellular domain of a prolactin receptor without cloning.

Furthermore, PCR primers can be designed to incorporate new restriction sites or other features such as termination codons at the ends of the gene segment to be amplified. This placement of restriction sites at the 5' and 3' ends of the amplified gene sequence allows for gene segments encoding EVPR or a fragment thereof to be custom designed for ligation other sequences and/or cloning sites in vectors.

PCR and other methods of amplification of RNA and/or DNA are well known in the art and can be used according to the present invention without undue experimentation, based on the teaching and guidance presented herein. Known methods of DNA or RNA amplification include, but are not limited to polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. patent Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188, to Mullis et al; 4,795,699 and 4,921,794 to Tabor et al; 5,142,033 to Ir is; 5,122,464 to Wilson et al; 5,091,310 to Innis; 5,066,584 to Gyllensten et al.; 4,889,818 to Gelfand et al; 4,994,370 to Silver et al; 4,766,067 to Biswas; 4,656,134 to Ringold; and Innis et al., eds., PCR Protocols: A Guide to Method and Applications) and RNA mediated amplification which uses anti-sense RNA to the target sequence as a template for double stranded DNA synthesis (U.S. patent No. 5,130,238 to Malek et al., with the trade name NASBA); and immuno-PCR which combines the use of DNA amplification with antibody labeling (Ruzicka et al., Science 260:487 (1993); Sano et al, Science 258:120 (1992); Sano et al, Biotechniques 9:1378 (1991)), the entire contents of which patents and reference are entirely incorporated herein by reference. In an analogous fashion, biologically active fragments of EVPRs (e.g. those of any of the Clone 19 or its isoforms) may be prepared as noted above with respect to the analogs of EVPRs. Suitable fragments of EVPRs are those which retain the Clone 19 activity and which can modulate or mediate the biological activity of JNK and/or NF- KB or other proteins associated with JNK and/or NF-κB directly or indirectly. Accordingly, EVPR fragments can be prepared which have a dominant-negative or a dominant-positive effect as noted above with respect to the analogs. It should be noted that these fragments represent a special class of the analogs of the invention, namely, they are defined portions of EVPRs derived from the full EVPR sequence (e.g., from that of any one of the Clone 19 or its isoforms), each such portion or fragment having any of the above-noted desired activities. Such fragment may be, e.g., a peptide.

Similarly, derivatives may be prepared by standard modifications of the side groups of one or more amino acid residues of EVPR, its analogs or fragments, or by linking of EVPR, its analogs or fragments, to another molecule e.g. an antibody, enzyme, receptor, a higher molecular weight polymeric material etc., as are well known in the art. Accordingly, "derivatives" as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or by linking the N- or C-terminal groups to other molecules, by means known in the art, and are included in the invention. Derivatives may have chemical moieties such as carbohydrate or phosphate residues, provided such a fraction has the same or higher biological activity as EVPRs.

An example of a higher molecular weight polymeric material is polyethylene glycol (PEG).

For example, derivatives may include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives or free amino groups of the amino acid residues formed with acyl moieties (e.g., alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group (for example that of seryl or threonyl residues) formed with acyl moieties. The term "derivatives" is intended to include only those derivatives that do not change one amino acid to another of the twenty commonly occurring natural amino acids.

EVPR is a protein or polypeptide, i.e. a sequence of amino acid residues. A polypeptide consisting of a larger sequence which includes the entire sequence of an EVPR, in accordance with the definitions herein, is intended to be included within the scope of such a polypeptide as long as the additions do not affect the basic and novel characteristics of the invention, i.e., if they either retain or increase the biological activity of EVPR or can be cleaved to leave a protein or polypeptide having the biological activity of EVPR. Thus, for example, the present invention is intended to include fusion proteins of EVPR with other amino acids or peptides.

Various recombinant cells such as prokaryotic cells, e.g., E. coli, or eukaryotic cells, such as yeast or insect cells can produce EVPR. Methods for constructing appropriate vectors, carrying DNA that codes for an EVPR and suitable for transforming (e.g., E. coli, mammalian cells and yeast cells), or infecting insect cells in order to produce a recombinant EVPR are well known in the art. See, for example, Ausubel et al., eds. "Current Protocols in Molecular Biology" Current Protocols, 1993; and Sambrook et al., eds. "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Spring Harbor Press, 1989. For the purposes of expression of EVPR proteins, DNA encoding an EVPR, their active fragments, analogs or derivatives, and the operably linked transcriptional and translational regulatory signals, are inserted into eukaryotic vectors which are capable of integrating the desired gene sequences into the host cell chromosome. In order to be able to select the cells which have stably integrated the introduced DNA into their chromosomes, one or more markers which allow for selection of host cells which contain the expression vector is used. The marker may provide for prototrophy to an auxo tropic host, biocide resistance, e.g., antibiotics, or resistance to heavy metals, such as copper, or the like. The selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by cotransfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

Said DNA molecule to be introduced into the cells of choice will preferably be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host. Preferred prokaryotic plasmids are derivatives of pBr322. Preferred eukaryotic vectors include BPV, vaccinia, SV40, 2-micron circle, etc., or their derivatives. Such plasmids and vectors are well known in the art (1-4, 19). Once the vector or DNA sequence containing the construct(s) has been prepared for expression, the expression vector may be introduced into an appropriate host cell by any of a variety of suitable means, such as transformation, transfection, lipofection, conjugation, protoplast fusion, electroporation, calcium phosphate precipitation, direct microinjection, etc.

Host cells to be used in this invention may be either prokaryotic or eukaryotic.

Preferred prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomvces, Pseudomonas, Salmonella, Serratia, etc. The most preferred prokaryotic host is E. coli.

Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli XI 776 (ATCC 31537), E. coli W3110 (F", lambda", phototropic (ATCC 27325). Under such conditions, the protein will not be glycosylated. The prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid. Eukaryotic hosts can also be used. Preferred eukaryotic hosts are mammalian cells, e.g., human, monkey, mouse and Chinese hamster ovary (CHO) cells, because they provide post-translational modifications to protein molecules including correct folding, correct disulfide bond formation, as well as glycosylation at correct sites. Also yeast cells and insect cells can carry out post-translational peptide modifications including high mannose glycosylation.

A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids, which can be utilized for production of the desired proteins in yeast and in insect cells. Yeast and insect cells recognize leader sequences on cloned mammalian gene products and secrete mature IL-18BP. After the introduction of the vector, the host cells are grown in a selective medium, which selects for the growth of vector-containing cells. Expression of the cloned gene sequence(s) results in the production of an EVPR, fusion proteins, or muteins or fragments thereof. The above-mentioned cloning, clone isolation, identification, characterization and sequencing procedures are described in more detail hereinafter in the Examples.

The expressed proteins are then isolated and purified by any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like, or by affinity chromatography, using, e.g., an anti-EVPR monoclonal antibodies immobilized on a gel matrix contained within a column. Crude preparations containing said recombinant EVPR are passed tlirough the column whereby EVPR will be bound to the column by the specific antibody, while the impurities will pass through. After washing, the protein is eluted from the gel under conditions usually employed for this purpose, i.e. at a high or a low pH, e.g. pH 11 or pH 2.

The invention further relates to vectors useful for expression of an EVPR or their derivatives in mammals and more specifically in humans. Vectors for short and long-term expression of genes in mammals are well known in the literature. Studies have shown that gene delivery to e.g., skeletal muscle, vascular smooth muscle and liver result in systemic levels of therapeutic proteins. Skeletal muscle is a useful target because of its large mass, vascularity and accessibility. However, other targets and particularly bone marrow precursors of immune cells have been used successfully.

Currently available vectors for expression of proteins in e.g., muscle include plasmid

DNA, liposomes, protein-DNA conjugates and vectors based on adenovirus, adeno- associated virus and herpes virus. Of these, vectors based on adeno-associated virus

(AAV) have been most successful with respect to duration and levels of gene expression and with respect to safety considerations (Kessler, P.D. 1996, Proc. Natl.

Acad. Sci. USA 93, 14082-14087).

Procedures for construction of an AAV-based vector have been described in detail (Snyder et al, 1996, Current Protocols in Human Genetics, Chapters 12.1.1-

12.1.17, John Wiley & Sons) and are incorporated into this patent. Briefly plasmid psub201, containing the wild-type AAV genome is cut with the restriction enzyme Xba I and ligated with a construct consisting of an efficient eukaryotic promoter, e.g., the cytomegalovirus promoter, a Kozak consensus sequence, a DNA sequence coding for an EVPR, or their muteins or fusion proteins or fragments thereof, a suitable 3' untranslated region and a polyadenylation signal, e.g., the polyadenylation signal of simian virus 40. The resulting recombinant plasmid is cotransfected with an helper AAV plasmid e.g., pAAV/Ad into mammalian cells e.g., human T293 cells. The cultures are then infected with adenovirus as a helper virus and culture supernatants are collected after 48-60 hours. The supernatants are fractionated by ammonium sulfate precipitation, purified on a CsCl density gradient, dialyzed and then heated at 56°C to destroy any adenovirus, whereas the resulting recombinant AAV, capable of expressing EVPR, or their muteins or fusion proteins remains stable at this step.

The new EVPRs, their analogs, fragments and derivatives thereof, have a number of possible uses, for example:

(i) EVPR, its analogs, fragments and derivatives thereof, may be used to modulate JNK and/or NF-κB activity in either of the inflammation, or the cell survival pathways as noted above. For example, if EVPR can prolong the duration of JNK activation and enhance JNK (Jun kinase) or p38 kinase or modulate NF-κB activation, both such EVPR effects leading to enhance such a Clone 19 effect when it would be desirable in anti-tumor, anti- or pro- inflammatory, anti-HIV applications, etc. In this case EVPR, its analogs, fragments or derivatives thereof, which modulate inflammation, or block the cell survival effect, may be introduced to the cells by standard procedures known per se. For example, when EVPR is entirely intracellular (as suspected) and should be introduced only into the cells where the Fas ligand or TNF or other cytotoxic protein effect, is desired, a system for specific introduction of this protein into the cells is necessary. One way of doing this is by creating a recombinant animal virus, e.g., one derived from Vaccinia, to the DNA of which the following two genes will be introduced: the gene encoding a ligand that binds to cell surface proteins specifically expressed by the cells, e.g., ones such as the AIDs (HIV) virus gpl20 protein which binds specifically to some cells (CD4 lymphocytes and related leukemias), or any other ligand that binds specifically to cells carrying a FAS-R or p55-R, such that the recombinant virus vector will be capable of binding such FAS-R or p55-R -carrying cells; and the gene encoding EVPR. 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 FAS-R or p55-R- carrying cell, following which the EVPR encoding sequence will be introduced into the cells via the virus, and once expressed in the cells, will result in modulation and/or mediation of JNK and/or NF-κB activity. Construction of such recombinant animal virus is by standard procedures (see for example, Sambrook et al., 1989). Another possibility is to introduce the sequences of EVPR (e.g., any one of the Clone 19 or its isoforms) in the form of oligonucleotides which can be absorbed by the cells and expressed therein.

(ii) EVPR, its analogs, fragments and derivatives, may be used to inhibit the Fas ligand or TNF or related protein effect, e.g., in cases such as tissue damage in septic shock, graft- vs. -host rejection, or acute hepatitis, in which it is desired to block the Fas ligand or TNF induced FAS-R or p55-R intracellular signalling or independent RIP effect, or other protein-mediated signalling and at the same time to increase the cell survival pathway. In this situation, it is possible, for example, to introduce into the cells, by standard procedures, oligonucleotides having the anti-sense coding sequence for EVPR, which would effectively block the translation of mRNAs encoding EVPR and thereby block its expression and lead to the inhibition of the Fas ligand-or TNF- or RIP-2 kinase or other protein- effect. Such oligonucleotides may be introduced into the cells using the above recombinant virus approach, the second sequence carried by the virus being the oligonucleotide sequence.

Likewise, as noted above, depending on the nature of the EVPR modulation and/or mediation of JNK and/or NF-κB activity, it may be possible by the ways of (i) and (ii) above to enhance or inliibit cell inflammation and survival pathways where desired.

Another possibility is to use antibodies specific for EVPR to inhibit its intracellular signalling activity. Yet another way of inhibiting the modulation and/or mediation of JNK and/or NF-κB activity is by the recently developed ribozyme approach. Ribozymes are catalytic RNA molecules that specifically cleave RNAs. Ribozymes may be engineered to cleave target RNAs of choice, e.g., the mRNAs encoding EVPR of the invention. Such ribozymes would have a sequence specific for EVPR mRNA and would be capable of interacting therewith (complementary binding) followed by cleavage of the mRNA, resulting in a decrease (or complete loss) in the expression of EVPR, the level of decreased expression being dependent upon the level of ribozyme expression in the target cell. To introduce ribozymes into the cells of choice (e.g., those carrying FAS- Ror p55-R), any suitable vector may be used, e.g., plasmid, animal virus (retrovirus) vectors, that are usually used for this purpose (see also (i) above, where the virus has, as second sequence, a cDNA encoding the ribozyme sequence of choice). (For reviews, methods etc. concerning 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). This approach is suitable when the Clone 19 modulation and/or mediation of JNK and/or NF-κB activity enhances cell cytotoxicity in situations when it is desired to block this cytotoxicity, or when the Clone 19 inhibits NF-κB activation in the same situation when it is desired to block this inhibition to increase such NF-κB activation, i.e. in both cases it is desired to increase cell survival as in (ii) above.

(iii) EVPR , its analogs, fragments or derivatives may also be used to isolate, identify and clone other proteins of the same class, involved in the modulation and/or mediation of JNK and/or NF-κB activity or to functionally related receptors or proteins, involved in the intracellular signalling process. In this application the above noted yeast two-hybrid system may be used, or there may be used a recently developed system employing non-stringent Southern hybridization followed by PCR cloning (Wilks et al, 1989). In the Wilks et al. publication, there is described the identification and cloning of two putative protein-tyrosine kinases by application of non-stringent southern hybridization followed by cloning by PCR based on the known sequence of the kinase motif, a conceived kinase sequence. This approach may be used, in accordance with the present invention using the sequence of EVPR to identify and clone those of related proteins capable of modulation and/or mediation of JNK and/or NF-κB activity.

(iv) Yet another approach for utilising EVPR , or its analogs, fragments or derivatives, of the invention is to use them in methods of affinity chromatography to isolate and identify other proteins or factors to which they are capable of binding, e.g., other proteins or factors involved in the intracellular signalling process. In this application, EVPR, its analogs, fragments or derivatives thereof, of the present invention, may be individually attached to affinity chromatography matrices and then brought into contact with cell extracts or isolated proteins or factors suspected of being involved in the intracellular signalling process. Following the affinity chromatography procedure, the other proteins or factors which bind to EVPR, or its analogs, fragments or derivatives thereof of the invention, can be eluted, isolated and characterised.

(v) As noted above, EVPR , or its analogs, fragments or derivatives thereof, of the invention may also be used as immunogens (antigens) to produce specific antibodies thereto. These antibodies may also be used for the puiposes of purification of EVPR (e.g., EVPR or any of its isoforms) either from cell extracts or from transformed cell lines producing EVPR, or its analogs or fragments. Further, these antibodies may be used for diagnostic purposes for identifying disorders related to abnormal functioning of the modulation and/or mediation of JNK and/or NF-κB activity, Fas ligand or TNF system, or independent activities, e.g., overactive or underactive Fas ligand- or TNF- induced cellular effects. Thus, should such disorders be related to a malfunctioning intracellular signalling system involving modulation and/or mediation of JNK and/or NF-κB activity, or various other above noted proteins or EVPR itself, such antibodies would serve as an important diagnostic tool. It should also be noted that the isolation, identification and characterisation of

EVPR of the invention may be performed using any of the well known standard screening procedures. For example, one of these screening procedures, the yeast two- hybrid procedure as is set forth herein above, was used to identify EVPR of the invention (besides various other new proteins of the above and below noted co-owned co-pending patent applications). Likewise as noted above and below, other procedures may be employed such as affinity chromatography, DNA hybridization procedures, etc. as are well known in the art, to isolate, identify and characterise the EVPR of the invention or to isolate, identify and characterise additional proteins, factors, receptors, etc. which are capable of binding to EVPRs of the invention. As set forth hereinabove, EVPR may be used to generate antibodies specific to

EVPRs and its isoforms. These antibodies or fragments thereof may be used as set forth hereinbelow in detail, it being understood that in these applications the antibodies or fragments thereof are those specific for EVPRs.

Based on the findings in accordance with the present invention that EVPR modulates and/or mediates JNK and/or NF-κB activity and can thus mediate/modulate

JNK and/or NF-κB activity in inflammation, cell death or cell survival pathways in ways that function independently or in conjunction with other proteins (e.g. Fas, p55-R,

MORT-1, MACH, Mch4, Gl and TRADD in cell death pathways, or with TRAF2 in cell survival pathways) it is of importance to design drugs which may enhance or inliibit the EVPR function, as desired and depending on which of these pathways are enhanced/inhibited by the EVPR. There are many diseases in which such drugs can be of great help. Amongst others, acute hepatitis in which the acute damage to the liver seems to reflect Fas ligand-mediated death of the liver cells; autoimmune-induced cell death such as the death of the β Langerhans cells of the pancreas, that results in diabetes; the death of cells in graft rejection (e.g., kidney, heart and liver); the death of oligodendrocytes in the brain in multiple sclerosis; and AIDS-inhibited T cell suicide which causes proliferation of the AIDS virus and hence the AIDS disease.

It is possible that EVPR or one or more of its possible isoforms may serve as "natural" inhibitors of JNK and/or NF-κB in one or more of the above pathways and these may thus be employed as the above noted specific inhibitors of JNK and/or NF- KB. Likewise, other substances such as peptides, organic compounds, antibodies, etc. may also be screened to obtain specific drugs, which are capable of inhibiting the EVPR modulation and/or mediation of JNK and/or NF-κB activity. A non-limiting example of how peptide inhibitors of the EVPR modulation and/or mediation of JNK and/or NF-κB activity would be designed and screened is based on previous studies on peptide inhibitors of ICE or ICE-like proteases, the substrate specificity of ICE and strategies for epitope analysis using peptide synthesis. The minimum requirement for efficient cleavage of peptide by ICE was found to involve four amino acids to the left of the cleavage site with a strong preference for aspartic acid in the PI position and with methylamine being sufficient to the right of the PI position (Sleath et al., 1990; Howard et al., 1991; Thornberry et al, 1992). Furthermore, the fluorogenic substrate peptide (a tetrapeptide), acetyl-Asp-Glu-Val- Asp-a-(4-methyl-coumaryl-7-amide) abbreviated Ac-DEVD-AMC, corresponds to a sequence in poly (ADP-ribose) polymerase (PARP) found to be cleaved in cells shortly after FAS-Rstimulation, as well as other apoptopic processes (Kaufmann, 1989; Kaufmann et al, 1993; Lazebnik et al., 1994), and is cleaved effectively by CPP32 (a member of the CED3/ICE protease family) and MACH proteases (and likewise also possibly by Gl proteases - see for example co-owned co-pending IL 120367).

As Asp in the PI position of the substrate appears to be important, tetrapeptides having Asp as the fourth amino acid residue and various combinations of amino acids in the first three residue positions can be rapidly screened for binding to the active site of the proteases using, for example, the method developed by Geysen (Geysen, 1985; Geysen et al., 1987) where a large number of peptides on solid supports were screened for specific interactions with antibodies. The binding of MACH proteases to specific peptides can be detected by a variety of well known detection methods within the skill of those in the art, such as radiolabeling of the Gl proteases, etc. This method of Geysen' s was shown to be capable of testing at least 4000 peptides each working day. In a similar way the exact region of homology which determines the modulation and/or mediation of JNK and/or NF-κB activity of EVPR can be elucidated and then peptides may be screened which can serve to block this modulation, e.g. peptides synthesized having a sequence similar to that of the modulation region or complementary thereto which can compete with natural EVPR for modulation and/or mediation of JNK and/or NF-κB activity. Drug or peptide inhibitors, which are capable of inhibiting inflammation induced by EVPR by inhibiting the EVPR modulation and/or mediation of JNK and/or NF-κB activity can be conjugated or complexed with molecules that facilitate entry into the cell. U.S. Patent 5,149,782 discloses conjugating a molecule to be transported across the cell membrane with a membrane blending agent such as fusogenic polypeptides, ion-channel forming polypeptides, other membrane polypeptides, and long chain fatty acids, e.g. myristic acid, palmitic acid. These membrane blending agents insert the molecular conjugates into the lipid bilayer of cellular membranes and facilitate their entry into the cytoplasm.

Low et al., U.S. Patent 5, 108,921, reviews available methods for transmembrane delivery of molecules such as, but not limited to, proteins and nucleic acids by the mechanism of receptor mediated endocytotic activity. These receptor systems include those recognizing galactose, mannose, mannose 6-phosphate, transferrin, asialoglycoprotein, transcobalamin (vitamin B12), α-2 macroglobulins, insulin and other peptide growth factors such as epidermal growth factor (EGF). Low et al. teaches that nutrient receptors, such as receptors for biotin and folate, can be advantageously used to enhance transport across the cell membrane due to the location and multiplicity of biotin and folate receptors on the membrane surfaces of most cells and the associated receptor mediated transmembrane transport processes. Thus, a complex formed between a compound to be delivered into the cytoplasm and a ligand, such as biotin or folate, is contacted with a cell membrane bearing biotin or folate receptors to initiate the receptor mediated trans-membrane transport mechanism and thereby permit entry of the desired compound into the cell. In addition, it is known in the art that fusing a desired peptide sequence with a leader/signal peptide sequence to create a "chimeric peptide" will enable such a "chimeric peptide" to be transported across the cell membrane into the cytoplasm.

As will be appreciated by those of skill in the art of peptides, the peptide inhibitors of the EVPR modulation and/or mediation of JNK and/or NF-κB activity according to the present invention is meant to include peptidomimetic drugs or inhibitors, which can also be rapidly screened for binding to EVPR to design perhaps more stable inhibitors.

It will also be appreciated that the same means for facilitating or enhancing the transport of peptide inhibitors across cell membranes as discussed above are also applicable to the EVPR or its isoforms themselves as well as other peptides and proteins which exert their effects intracellularly.

As regards the antibodies mentioned herein throughout, the term "antibody" is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments thereof 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 to antigens, which populations contains substantially similar epitope binding sites. MAbs may be obtained by methods known to those skilled in the art. See, for example Kohler and Milstein, Nature, 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al., eds., Harlow and Lane ANTIBODIES : A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al, eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience N.Y., (1992-1996), the contents of which references are incorporated entirely herein by reference. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclass thereof. A hybridoma producing a mAb of the present invention may be cultivated in vitro, in situ or in vivo. Production of high titers of mAbs in vivo or in situ makes this the presently preferred method of production.

Chimeric antibodies are molecules of which different portions are derived from different animal species, such as those having the variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proc. Natl. Acad. Sci. USA 81 :3273-3277 (1984); Morrison et al, Proc. Natl. Acad. Sci. USA 81 :6851-6855 (1984); Boulianne et al, Nature 312:643-646 (1984); Cabilly et al., European Patent Application 125023 (published November 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); Robinson et al, International Patent Application No. WO8702671 (published May 7, 1987); Liu et al., Proc. Natl. Acad. Sci USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci 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 entirely incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody that recognises unique determinants generally associated with the antigen-binding site of an antibody. An Id antibody can be prepared by immunising an animal of the same species and genetic type (e.g. mouse strain) as the source of the mAb to which an anti-Id is being prepared. The immunised animal will recognise and respond to the idiotypic determinants of the immunising antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example, U.S. Patent No. 4,699,880, which is herein entirely incorporated by reference. The anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody. The anti- anti-Id may be epitopically identical to the original mAb, which induced the anti-Id. Thus, by using antibodies to the idiotypic determinants of a mAb, it is possible to identify other clones expressing antibodies of identical specificity. Accordingly, mAbs generated against EVPR s, analogs, fragments or derivatives thereof, of the present invention may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice. Spleen cells from such immunised mice are used to produce anti-Id hybridomas secreting anti-Id mAbs. Further, the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunise additional 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 EVPR, or analogs, fragments and derivatives thereof.

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

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

It will be appreciated that Fab and F(ab')₂ and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of EVPR according to the methods disclosed herein 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 capable of specifically reacting with the molecule to thereby bind the molecule to the antibody. The term "epitope" is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognised by that antibody. Epitopes or "antigenic determinants" usually consist of chemically active surface groupings 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 a portion of a molecule capable of being bound by an antibody, which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen. An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which may be evoked by other antigens. The antibodies, including fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect EVPR in a sample or to detect presence of cells, which express EVPR of the present invention. This can be accomplished by immunofluorescence techniques employing a fluorescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.

The antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of EVPR of the present invention. In situ detection may be accomplished by removing a histological specimen from a patient, and providing the labelled antibody of the present invention to such a specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labelled antibody (or fragment) to a biological sample. Through the use of such a procedure, it is possible to determine not only the presence of EVPR, but also its distribution on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Such assays for EVPR of the present invention typically comprises incubating 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 tissue culture, in the presence of a detectably labeled antibody capable of identifying EVPR, and detecting the antibody by any of a number of techniques well known in the art.

The biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins. The support or carrier may then be washed with suitable buffers followed by treatment with a detectably labeled antibody in accordance with the present invention, as noted above. The solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody. The amount of bound label on said solid support or carrier may then be detected by conventional means. By "solid phase support", "solid phase carrier", "solid support", "solid carrier",

"support" or "carrier" is intended 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 be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody. Thus, the support or carrier configuration may be spherical, as in a bead, cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Preferred supports or carriers include polystyrene beads. Those skilled in the art will know may other suitable earners for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.

The binding activity of a given lot of antibody, of the invention as noted above, may be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

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

One of the ways in which an antibody in accordance with the present invention can be detectably labeled is by linldng the same to an enzyme and used in an enzyme immunoassay (EIA). This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, alate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomeras, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholin-esterase. The detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of other immunoassays. For example, by radioactive labelling the antibodies or antibody fragments, it is possible to detect R-PTPase tlirough the use of a radioimmunoassay (RIA). A good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, 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., incorporated by reference herein. The radioactive isotope can be detected by such means as the use of a g counter or a scintillation counter or by autoradiography.

It is also possible to label an antibody in accordance with the present invention with a fluorescent compound. When the fluorescently labelled antibody is exposed to light of the proper wavelength, its presence can be then detected due to fluorescence. Among the most commonly used fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine.

The antibody can also be detectably labelled using fluorescence emitting metals such as 152E, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamme pentaacetic acid (ETPA).

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

Likewise, a bioluminescent compound may be used to label the antibody of the present invention. Bioluminescence is a type of chemiluminescence found in biological systems in which 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 purposes of labelling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labelled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labelled antibody. Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody- antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labelled antibody (which functions as a "reporter molecule"). After a second incubation period to permit the labelled antibody to complex with the antigen bound to the solid support or carrier through the unlabeled antibody, the solid support or carrier is washed a second time to remove the unreacted labelled antibody. In another type of "sandwich" assay, which may also be useful with the antigens of the present invention, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labelled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labelled antibody. The presence of labelled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labelled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilised. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted-labelled antibody. The determination of labelled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays. EVPRs of the invention may be produced by any standard recombinant DNA procedure (see for example, Sambrook, et al., 1989 and Ansabel et al., 1987-1995, supra) in which suitable eukaryotic or prokaryotic host cells well known in the art are transformed by appropriate eukaryotic or prokaryotic vectors containing the sequences encoding for the proteins. Accordingly, the present invention also concerns such expression vectors and transformed hosts for the production of the proteins of the invention. As mentioned above, these proteins also include their biologically active analogs, fragments and derivatives, and thus the vectors encoding them also include vectors encoding analogs and fragments of these proteins, and the transformed hosts include those producing such analogs and fragments. The derivatives of these proteins, produced by the transformed hosts, are the derivatives produced by standard modification of the proteins or their analogs or fragments.

The present invention also relates to pharmaceutical compositions comprising recombinant animal virus vectors encoding EVPRs, which vector also encodes a virus surface protein capable of binding specific target cell (e.g., cancer cells) surface proteins to direct the insertion of EVPR sequences into the cells. Further pharmaceutical compositions of the invention comprises as the active ingredient (a) an oligonucleotide sequence encoding an anti-sense sequence of EVPR sequence, or (b) drugs that block the EVPR modulation and/or mediation of JNK and/or NF-κB activity.

Pharmaceutical compositions according to the present invention include a sufficient amount of the active ingredient to achieve its intended purpose. The dosage will also depend on the body weight of the patient treated, as well as on the manner of administration. In addition, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically and which can stabilize such preparations for administration to the subject in need thereof as well known to those of skill in the art.

EVPR and its isoforms or isotypes are suspected to be expressed in different tissues at markedly different levels and apparently also with different patterns of isotypes in an analogous fashion to the expression of various other proteins involved in the intracellular signaling pathways as indicated in the above listed co-owned co- pending patent applications. These differences may possibly contribute to the tissue- specific features of response to the Fas ligand and TNF. As in the case of other CED3/ICE homologs (Wang et al., 1994; Alnemri et al., 1995), the present inventors have previously shown (in the above mentioned patent applications) that MACH isoforms that contain incomplete CED3/ICE regions (e.g., MACHα3) are found to have an inhibitory effect on the activity of co-expressed MACHαl or MACHα2 molecules; they are also found to block death induction by FAS-R and p55-R. Expression of such inhibitory isoforms in cells may constitute a mechanism of cellular self-protection against FAS-R and TNF-mediated cytotoxicity. A similar inhibitory effect of at least some Gl isoforms is also suspected (Gl being a recently isolated new Mch4- and possibly MACH- binding protein, and also MORT-1 -binding protein that has MORT MODULES and a protease domain - see co-owned co-pending IL 120367). The wide heterogeneity of MACH isoforms, and likewise the suspected, analogous heterogeneity of Gl isoforms, which greatly exceeds that observed for any of the other proteases of the CED3/ICE family, should allow a particularly fine tuning of the function of the active MACH isoforms, and by analogy also the active Gl isoforms. Hence, as noted above, EVPRs or possible isoforms may have varying effects in different tissues as regards their modulation and/or mediation of JNK and/or NF-κB activity and their influence thereby on the balance between activation of cell death or cell survival pathways. It is also possible that some of the possible EVPR isoforms serve other functions. For example, EVPR or some EVPR isoforms may also act as docking sites for molecules that are involved in other, non-cytotoxic effects of Fas and TNF receptors. Due to the unique ability of FAS-R and TNF receptors to cause inflammation, cell death, as well as the ability of the TNF receptors to trigger other tissue-damaging activities, aberrations in the function of these receptors could be particularly deleterious to the organism. Indeed, both excessive and deficient functioning of these receptors have been shown to contribute to pathological manifestations of various diseases (Vassalli, 1992; Nagata and Golstein, 1995). Identifying the molecules that participate in the signaling activity of the receptors, and finding ways to modulate the activity of these molecules, could direct new therapeutic approaches. Other aspects of the invention will be apparent from the following examples.

The invention will now be described in more detail in the following non-limiting examples and the accompanying drawings:

It should also be noted that the procedures of: i) two-hybrid screen and two-hybrid β-galactosidase expression test; (ii) induced expression, metabolic labeling and immunoprecipitation of proteins; (iii) in vitro binding; (iv) assessment of the cytotoxicity; and (v) Northern and sequence analyses, as well as other procedures used in the following Examples have been detailed in previous publications by the present inventors in respect of other intracellular signalling proteins and pathways (see, for example, Boldin et al., 1995a, 1995b, and Boldin et al. 1996). These procedures also appear in detail in the co-owned co-pending Israel Application Nos. 114615, 114986, 115319, 116588, 117932, and 120367 as well as the corresponding PCT application No. PCT/US96/10521). Accordingly, the full disclosures of all these publications and patent applications are included herein in their entirety and at least as far as the detailed experimental procedures are concerned. EXAMPLES

Example 1: Cloning of Clone 19

Using the two hybrid screening procedure with MORT-1 as bait, an oligodT and random primed cDNA library prepared from 11.5 days old mouse embryos cloned into the pGAD GH vector was screened and a murine clone 19 cDNA was identified, isolated and characterized.

The original cDNA of clone 19 (clone 19or for original, herein) was cloned by the two hybrid screening method. The so-isolated clone 19-cDNA insert was sequenced using standard DNA sequencing procedures and found to contain 517 nucleotides encoding a protein with a deduced 170 amino acids sequence. The amino acid sequence of clone 19 was deduced from the DNA sequence using DNA Strider VI .2 software.

An additional clone 19 cDNA was cloned by PCR from the above noted library by using appropriate PCR-primers, designed by OLIGO5™ software. A sense primer encoding a sequence from the 3' end sequence of clone 19or and an antisense primer derived from the pGAD GH vector sequences were used. This cDNA clone was found to contain an additional 260bp at the 3' end of the sequence of clone 19or. Altogether the two cDNA clones encode a protein with a deduced sequence of about 260 amino acids. A cDNA clone comprising nucleotides 1 to 777, which encode the 260 amino acids was constructed, and is herein-designated clone 19ex, for extended, or clone 19a. The nucleotide sequence of clone 19a was compared to sequences found in the databases and found to exhibit homology to published murine and human EST sequences. A murine EST (accession number 1397922) was identified which had 93.5% identity with clone 19 in their first 200bp but to differ thereafter. Two human sequences (accession number Y13871, Zuehlke C et all, DNA Seq. 10, pp. 1-6, 1999, and accession number AA327964, Adams, M.D et all, Nature, 377, pp.3-174, 1995) display 76.9% and 79% identity respectively to the sequence of murine clone 19a. An additional murine EST (accession number 933841) was found to be 79.3% identical to the 3' end of the human EST sequences Y13871. A variant of clone 19 was cloned by PCR after reverse transcription of RNA extracted from mouse spleen. The reverse transcriptase reaction was performed with an oligo (dT) primer and the AMV reverse transcriptase (available from Promega) according to the manufacturer's instructions. The PCR reaction was carried out using sense oligonucleotide primers from clone 19 ex and antisense primers derived from the EST 933841 sequence, and the resulting PCR product was cloned into a vector and sequenced. This variant of clone 19 designated clone 19c was found to display 68.3% identity with the human EST sequence Y13871.

The murine EST clone #1397922 was obtained from Research Genetics (Huntsville, AL) and fully sequenced. Full sequencing of the EST resulted in a different sequence from the published sequence and was identified as an additional variant of clone 19 and designated clone 19d.

The sequences of the murine clone 19 variants clone 19a, clone 19c and clone

19d is shown in Figure 1. The alignment of the deduced amino acid sequence of the murine clone 19 variants with the ESTs found in the public databases is shown in figure

2.

Example 2: Cloning of human Clone 19 isoforms

The human clone 19 was cloned from a human leukemia library (5'-STRETCH PLUS cDNA library, Clontech Laboratories, Inc., Palo Alto, Ca, USA) derived from the Jurkat T-cell line. The library was screened under stringent conditions using a 480 bp probe amplified by PCR from the above mentioned library using sense

(5 ' CGCAGTGGACTGTGTCATTGACCT 3 ' ) and antisense

(5 ' CAAGGCTCGTGGTGGACAAGGTGA 3 ' ) oligonucleotide primers derived from the sequence of clone Y13871. The probe was labelled using a random-priming kit (Boehringer Mannheim). The two human clone 19 variats cloned from the library were designated human clone 19a and clone 19b. The sequence of the two human clone 19 variants is shown in Figure 1. The sequence of the two clones is practically identical starting with nucleotide 399 of clone 19a up to nucleotide 3028, and nucleotide 111 of clone 19b up to nucleotide 2811, clone 19b having an additional T in the region of nucleotides 2488-

2494. The aligmnent of the deduced amino acid sequence of the human clone 19 variants with the murine clone 19 variants and the ESTs found in the public databases is shown in figure 2. In this figure the comparison starts with the ATG which for the murine clones 19a and 19c is nucleotide 71, and nucleotide 86 for murine clone 19d (according to fig. 1). It should be noted that none of the EVPR sequences contains a death domain.

Example 3: Interactions of clone 19 in yeast two-hybrid tests

A two-hybrid test in yeast was performed in order to evaluate the interaction of clone 19 with other proteins that mediate the TNF-induced signalling cascade. The two-hybrid test was performed by cloning clone 19or and clone 19ex into the GAD vector used for cloning the library (the "prey" vector) or into the GBT vector used for expressing the "bait" used for screening of the library (Table II). The two-hybrid tests in yeast revealed that when expressed from the GAD "prey" vector, clone 19or and clone 19ex strongly interact with themselves, with the full length MORT1 as well as with the full length and Death Domains (DD) of RIP, TRADD, p55-R, and FAS-R (Table II). LPR- like mutations which disrupt the structure of the FAS-R death domain (Varfolomeev et al, J. Exp. Med., 183, 1271-1275, 1995) were introduced into the DD of RIP, p55-R, and Fas. These mutations abrogated binding of the mutant proteins with clone 19 in the two-hybrid test (Table II). As can be seen from Table II, Clone 19 binds full length CASH Alfa and beta, suggesting it binds to the death effector domains (DED) of those molecules as shown. Clone 19 also strongly binds to TRAF 1, TRAF 2 and TRAF 6. In these two-hybrid tests clone 19 did not bind to caspases 1, 2, and 10 to NIK, RIP2, p75TNFR, NGF, and Apaf 1, nor did it bind to the non-relevant proteins lamin and SNF used as controls.

It is interesting to note that clone 19 is capable of binding to a number of death domain-containing proteins, despite the fact that it does not contain a death domain itself.

Table II: Two-hybrid tests of clone 19 interactions

* Color appearance < 1 hour +++ ; 1 - 4 hours ++ ; 4 hours - overnight + ; No interaction - ; ND= not done Example 4: Functional characterisation of clone 19 a) Cytotoxicity: The initial functional characterisation of clone 19 indicated that transient cotransfection of HEK 293 -T cells with clone 19or (and clone 19ex) did not induce cytotoxicity, nor did it affect cytotoxicity induced by co-expression of the p55TNFR, FAS-R, TRADD, MORT, RIP, and MACH in HEK-293T cells (not shown). b) NF-κB activation: In order to examine whether clone 19 is capable of interfering with NF-κB activation in cell culture, clone 19or (and clone 19ex) was overexpressed with known inducers of NF-κB activation. HEK-293T cells were transiently cotransfected with TRADD and with p55TNFR and with a reporter plasmid comprised of the luciferase gene under control of the HIV-LTR minimal promoter. This reporter plasmid carries a NF-κB responsive element and is used as an indicator of NF-κB induction. Overexpression of clone 19 resulted in induction of NF-κB to levels similar to those induced by TRAF2. NF-κB induction by TRAF2 is significant, but not high. Clone 19or (and clone 19ex) also potentiated NF-κB induction by TRADD by 10-25 % (Figure 3). c) JNK activation: In order to examine the effect of Clone 19 on TNF induced JNK activation, Clone 19ex was transiently co-transfected in TNF treated 293T cells and Hela-FAS-R cells, which constitutively express FAS-R, together with a HA- JNKl - expressing plasmid. JNK1 was then immunoprecipitated via its N-terminal HA-tag and its ability to phosphorylate bacterially produced, purified GST-c-Jun was determined by

P-mcorporation m an in vitro kinase assay. Reaction products were analysed by SDS-

PAGE as shown in figure 4. JNKl immunoprecipitated from cellular lysates of HEK-

293T cells and Hela-FAS-R cells, transfected with the HA- JNKl -expressing plasmid together with the Clone 19ex expressing plasmid for the indicated period of time were used in the in vitro kinase assay with purified GST-c-Jun as a substrate. It was found that upon treatment of transfected cells with TNF (lOOOOU/ml) clone 19ex could prolong the duration of JNK activation in Hela-FAS-R cells and enhance JNK activity in 293T cells (Figure 4). As clone 19or is able to trigger JNK activation by itself, TNF does not significantly potentiate clone 19or induced JNK activation in cells cotransfected with clone 19or (Figure 5). d) Modulation of RIP-2 activity: When cotransfected with RIP-2 (IBK7B1/B1 kinase), clone 19ex and clone 19or led to a two and three fold reduction in NF-κB induction by RIP-2 respectively (Figure 6). Clone 19or also effectively blocks RIP-2-induced JNK activation (Figure 7), suggesting that Clone 19 inhibits RIP-2 kinase signalling. Clone 19or induced JNK activation most likely occurs downstream of RIP-2 in the signalling pathway, since coexpression of Clone 19or and RIP-2 results in inhibition of JNK activation to the levels of JNK activation similar to those induced by Clone 19or alone. e) Inhibition by dominant-negative TRAF-2: TRAF-2 dominant negative (DN) mutant downregulates clone 19or and clone 19ex TNF induced JNK activation (Figure 5, lower panel), suggesting that clone 19 may act either at the same level or upstream to TRAF-2 in the signalling pathway. TRAF2 dominant-negative mutant overexpression may inhibit the activity of molecules acting upstream to it.

Example 5: Northern blot analysis of clone 19 mRNA expression

The expression of clone 19 was analysed in embryonic and adult mouse tissues using mouse multiple tissue and embryo tissue blots containing polyA + RNA (cat # 7762-1 and cat # 7763-1 respectively, Clontech Laboratories, Inc, Palo Alto, CA). The blots were hybridised with the 517-bp cDNA insert of clone 19or probe. Northern blot analysis of embryonic tissue revealed a major transcript of approximately 4.8-5.0 kb for clone 19 which was not detectable on day 7 and appeared beginning on day 11. In adult tissues clone 19 was found to be expressed in the testis and liver as well as in brain and heart, but not in spleen and skeletal muscle. A weak signal was detected in lung and kidney (Figure 8). In the liver a minor transcript of about 1.8-2.0 kb can be seen.

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Claims

1. A DNA sequence encoding a MORT-1 interacting protein, herein designated EVPR, or isoforms, fragments, analogs or derivatives thereof, said protein capable of modulating NF-κB activation and of JNK activity, and lacking a death domain.

2. A DNA sequence according to claim 1, selected from the group i) a cDNA sequence of the herein designated murine clone 19a (depicted in fig. 1), j) a cDNA sequence of the herein designated murine clone 19c (depicted in fig. 1), k) a cDNA sequence of the herein designated murine clone 19d (depicted in fig- 1),

1) a cDNA sequence of the herein designated human clone 19a (depicted in fig. 1), m) a cDNA sequence of the herein designated human clone 19b (depicted in fig. 1), n) a fragment of sequence (a)-(e), o) a DNA sequence capable of hybridization to a sequence of (a)-(f) under stringent conditions, p) a DNA sequence which is degenerate as a result of the genetic code to the DNA sequences defined in (a)-(g).

3. A DNA sequence according to claim 2, comprising nucleotides 111 to 2493 of human clone 19b depicted in fig. 1.

4. A vector comprising a DNA sequence according to any one of claims 1-3.

5. A vector according to claim 4, capable of being expressed in a eukaryotic host cell.

6. A vector according to claim 4, capable of being expressed in a prokaryotic host cell.

7. Transformed eukaryotic or prokaryotic host cells containing a vector according to any one of claims 4-6.

8. A MORT-1 interacting protein, herein designated EVPR, isoforms, fragments, analogs or derivatives thereof, capable of modulating NF-κB activation and of JNK activity, said protein lacking a death domain.

9. A protein according to claim 8, encoded by a DNA sequence according to any one of claims 1 to 3.

10. A protein according to claim 9, encoded by at least nucleotides 111 to 2493 of human clone 19b depicted in fig. 1.

11. A method for producing a protein, isoform, fragment, analog or derivative thereof, according to any one of claims 8 to 10, comprising growing a transformed host cell according to claim 7 under conditions suitable for expression of said protein, isoform, fragment, analog or derivative thereof, effecting post-translational modifications, as necessary, for obtaining said protein, isoform, fragment, analog or derivative thereof, and isolating said protein, isoform, fragment, analog or derivative thereof.

12. Antibodies or active fragments or derivatives thereof, specific for a MORT-1 interacting protein, herein designated EVPR, isoforms, fragments, analogs or derivatives thereof, capable of modulating NF-KB activation and of JNK activity, said protein lacking a death domain.

13. A method for the modulation in cells of the activity of NF-κB or JNK, said method comprising treating said cells by introducing into said cells one or more of a protein, isoform, analog, fragment or derivative thereof, according to any one of claims 8 to 10, in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more protein, isoform, analog, fragment or derivative thereof in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.

14. A method according to claim 13, wherein said treating of cells comprises introducing into said cells a DNA sequence encoding said protein, isoform, fragment, analog or derivative in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells.

15. A method according to claim 13 or 14 wherein said treating of said cells is by transfection of said cells with a recombinant animal virus vector comprising the steps of :

(a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of said cells to be treated and a second sequence encoding a protein selected from the said protein, isoforms, analogs, fragments and derivatives according to any one of claims 8-10, that when expressed in said cells is capable of modulating/mediating the activity of NF-κB or any other intracellular signaling activity modulated/mediated by TRAF2 or other said molecules; and (b) infecting said cells with said vector of (a).

16. A pharmaceutical composition for the modulation of the activity of NF-κB or JNK comprising, as active ingredient at least one protein according to any one of claims 8-11, its biologically active isoforms, fragments, analogs, derivatives or mixtures thereof.

17. A pharmaceutical composition for modulating the activity of NF-kB or JNK comprising, as active ingredient, a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding at least one protein, isoform, active fragment or analog, according to any one of claims 8-11.