CZ20003421A3 - DNA sequence encoding protein associated with RIP, protein encoded by this sequence and methods of use thereof - Google Patents

Abstract

The solution describes a novel protein mediating or mediating intercellular RIP activity in signal transduction pathways leading to inflammation, cell survival or cell death. It further describes DNA encoding said protein and its use.

Description

Technical field

The present invention generally relates to receptors from the TGF / NGF superfamily and control of their biological functions. TGF / NGF receptor superfamily includes receptors such as p55 and p75 tumor necrosis factor receptors (TNF-R, herein referred to as p55-R and p75-R), and FAS ligand receptors (also referred to as FAS / APO or FAS-R, and herein referred to as FAS-R) and others.

More specifically, the present invention relates to novel proteins that bind to other proteins that bind directly or indirectly to members of the TNF / NGF receptor family and to other intracellular modulating proteins.

More specifically, the invention relates to one such protein referred to herein as RAP-2 (RIP-associated protein 2) and its isoforms, fragments and derivatives thereof, as well as proteins that bind to RAP-2.

RAP-2 binds to RIP (a receptor interacting protein) and may affect the function of RIP and thus also affect or mediate, directly or indirectly, the function of other proteins that bind directly or indirectly to RIP. RAP-2 binding proteins are modulators / mediators of RAP-2 function.

BACKGROUND OF THE INVENTION

Tumor necrosis factor (TNF-α) and lymphotoxin (TNF-β) (hereinafter TNF-α and TNF-β) are multifunctional pro-inflammatory cytokines produced primarily in mononuclear phagocytes that have many different effects on cells (Wallach,

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D, 1986, in: Interferon 7 (Ion Gresser, ed.), Pp. 83-122, Academic Press, London; and Beutler and Cerami, 1987. Both TNF-α and TNF-β initiate their action by binding to specific cell surface receptors. Some of their effects are likely to be beneficial to the body: they can, for example, destroy tumor cells or cells infected with viruses and can enhance the antibacterial activity of granulocytes. In this way, TNF contributes to the body's defense against tumors and infectious agents and contributes to wound healing. Therefore, TNF can be used as an antitumor agent and in this application binds to its receptors on the surface of tumor cells and thus triggers events leading to tumor cell death. TNF can also be used as an antiinfective.

However, both TNF-α and TNF-β also have deleterious effects. There is evidence that overproduction of TNF-α can play a major pathogenetic role in several diseases. For example, the effects of TNF-α on the vascular system are known to be a major cause of septic shock symptoms (Tracey et al., 1986). In some diseases, TNF may cause disproportionate weight loss (cachexia) by suppressing adipocyte activity and causing anorexia, and therefore TNF-α was also referred to as cachexin. It has also been reported to be a mediator of tissue damage in rheumatic diseases (Beutler and Cerami, 1987) and to be a major mediator of damage observed in graft versus host reactions (Piquet et al., 1987). Furthermore, TNF is known to be involved in inflammatory processes and many other diseases.

The above biological effects of TNF mediate and / or initiate two different, independently expressed receptors, p55 and p75 TNF-R, which specifically bind both TNF-α and TNF-β. These two receptors have structurally different intracellular domains, suggesting that signal transduction is different (see Hohmann et al., 1989; Engelmann et al., 1990; Brockhaus et al., 1990; Loetscher et al., 1990; Schall et al., 1990; Nophar et al., 1990; Smith et al., 1990; and Heller et al., 1990). However, cellular mechanisms, such as various proteins and perhaps other factors involved in intracellular signal transduction from p55 and p75 TNF-R, are not yet known. This intracellular signal transduction, which occurs after ligand binding, i.e., TNF (and / or β) to the receptor, is responsible for triggering a cascade of responses that ultimately lead to the observed cell response to TNF.

The above-mentioned cytocidal effect of TNF is mainly triggered by p55 TNF-R in most of the cells studied to date. Antibodies against the extracellular (ligand binding domain) pp55 TNF-R themselves can trigger a cytocidal effect (see EP 412486), which correlates with the efficacy of receptor cross-linking by antibodies, which is probably the first step in intracellular signal transduction. Furthermore, mutation studies (Brakebusch et al., 1992; Tartaglia et al., 1993) have shown that the biological function of p55 TNFR depends on the integrity of its intracellular domain. Therefore, it was hypothesized that the initiation of intracellular signaling leading to the cytocidal effect of TNF occurs due to the association of two or more intracellular domains of p55 TNF-R. In addition, TNF (a and β) occur as homotrimers, and it is therefore believed that the induction of intracellular signaling by p55 TNF-R occurs due to the ability of TNF to bind to and cause cross-linking of receptor molecules, i.e., receptor aggregation.

Another member of the TGF / NGF receptor superfamily is the FAS receptor (FAS-R), also referred to as the FAS antigen, a cell surface protein expressed in many tissues that exhibits homology to many surface cell receptors, including TNF-R and NGF- R. FAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991) and appears to serve as a negative selection factor for autoreactive T cells, i.

that during maturation of T cells, FAS-R mediates the apoptotic death of T cells recognizing self antigens. FAS-R gene (lpr) mutations have also been found to cause lymphoproliferative diseases in mice that mimic human autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The FAS-R ligand appears to be a cell surface molecule present, inter alia, on Tkiiier lymphocytes (or cytotoxic T lymphocytes - CTLs), therefore, when such CTLs contact FAS-R-bearing cells, CTLs can induce apoptosis of FAS- R. FAS-R-specific monoclonal antibodies were also prepared and these monoclonal antibodies were able to induce apoptosis of FAS-R-bearing cells, including mouse cells transformed with cDNA encoding human FAS-R (Itoh et al., 1991).

The inventors have discovered many approaches (see European Patent Application Nos. EP 186833, EP 308378, EP 398327 and EP 412486) for controlling the deleterious effects of TNF by inhibiting TNF binding to their receptors using anti-TNF antibodies or soluble TNF receptors that compete with TNF binding to TNFR cell membranes. In addition, since the binding of TNF to their receptors is necessary for TNF-induced effects on the cell, the inventors (see, for example, EP 568925) attempted to modulate the effect of TNF by modulating TNF-R activity.

Briefly, EP 568925 relates to a method for modulating signal transduction and / or cleavage of TNF-R, in which peptides or other molecules can interact with the receptor itself or with receptor-interacting effector proteins, which • modulates the normal function of TNF-R. EP 568925 describes the construction and characterization of various p55 TNF-R mutants having mutations in the extracellular, transmembrane, and intracellular domains of p55 TNF-R. In this way, regions in the p55 TNF-R domains that are essential for receptor function, i.e., ligand binding (TNF) and for subsequent signal transduction and intracellular signaling, ultimately leading to the observed effects of TNF on cells, have been identified. In addition, a number of approaches for isolating and identifying proteins, peptides, or other factors that can bind to different regions in the above TNF-R domains, where these proteins, peptides, and other factors may be involved in regulating or modulating TNF- R. Many procedures for isolating and cloning DNA sequences encoding such proteins and peptides; for the construction of expression vectors for the production of these proteins and peptides; and for the preparation of antibodies or fragments thereof that interact with TNF-R or the aforementioned proteins and peptides that bind to different regions of TNF-R is also disclosed in EP 568925. However, EP 568925 does not specify specific proteins and peptides that it binds to the intracellular domains of TNF-R (e.g., p55 TNF-R), nor does it disclose a yeast two-hybrid procedure for the isolation and identification of such proteins or peptides that bind to the intracellular domains of TNF-R. Also, EP 568925 does not disclose proteins or peptides that bind to the intracellular domains of FAS-R.

Although tumor necrosis factor (TNF) receptors and structurally related FAS-Rs are known to trigger, in cells - after stimulation with ligands produced by leukocytes, destructive activities that lead to their own damage, this trigger mechanism is still little known. Mutational studies show that FAS-R and p55 TNF receptor (p55-R) signaling leading to cytotoxicity are involved in different regions in their tftf · tftftftf · intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993). These regions (death domains) have sequence similarities. The deadly domains FAS-R and p55-R tend to self-associate. This self-association clearly promotes receptor aggregation, which is necessary to initiate signaling (see Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995), and high levels of receptor expression can trigger signaling that is not dependent on ligands (Boldin et al., 1995).

Like other events induced by receptors, cell death induction of TNF receptors and FAS-R occurs through a series of protein-protein interactions that lead from ligand binding to the receptor to eventual activation of enzymatic effector functions that were evaluated in non-enzymatic protein-protein interaction studies initiating signaling for cell death: binding of trimeric TNF or FAS-R ligand molecules to these receptors, resulting in the interaction of their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993) increase the likelihood of spontaneous association of motifs (deadly domains (Boldin et al, 1995a) and induce the binding of two cytoplasmic proteins (which also bind to one another) to the intracellular domains of the receptors - MORT-1 (or FAD) to FAS_R (Boldin et al., 1995b; Chinnaiyan et al., 1995; Kischkel et al., 1995) and TRADD on p55-R (Hsu et al., 1995; Hsu et al., 1996). Three proteins that bind to the intracellular domain of FAS-R and p55-R in the death domain region involved in the induction of cell death via receptors, through the hetero-association of homologous regions, and which, independently, are also capable of triggering cell death have been identified One of these is the MORT-1 protein (Boldin et al., 1995b), also known as FADD (Chinnaiyan et al., 1995), which

specifically binds to FAS-R. The second is TRADD (see also Hsu et al., 1995, 1996), which binds to p55-R and the third is RIP (see also Stanger et al., 1995), which binds to both FAS-R and p55R . In addition to binding to FAS-R and p55-R, these proteins can also bind to each other, allowing functional communication between FAS-R and p55-R. This binding occurs through a conserved motif sequence, a death domain module that is common to receptors and their associated proteins. Furthermore, although it has been shown in the yeast two-hybrid assay that MORT-1 binds spontaneously to FAS-R on mammalian cells, this binding only occurs after receptor stimulation, suggesting that MORT-1 is involved in initiating FAS-R signaling events. MORT-1 does not contain any sequence motif characteristic of enzymatic activity and therefore its ability to trigger cell death does not appear to lie in the intrinsic activity of MORT-1 itself, but rather in the activation of some other proteins to which MORT-1 binds and which acts further in the signaling cascade. Cell expression of MORT-1 mutants lacking the N-terminal portion of the molecule has been shown to block cytotoxic induction by FAS-APO1 (FAS-R) or p55-R (Hsu et al., 1996; Chinnaiyan et al.,

1996), suggesting that this N-terminal region transmits a signal for the cytocidal effect of both receptors through protein-protein interactions.

Therefore, the death domain motifs of the p55-R and FAS-R receptors, as well as their three associated proteins MORT-1, RIP and TRADD, appear to be sites of protein-protein interactions. The three proteins, MORT-1, RIP and TRADD, interact with the intracellular domains of p55-R and FAS-R through the binding of their lethal domains to receptor domains, and their domains also self-associate (although MORT-1 is different in that its deadly domain is not subject to spontaneous association). In addition, MORT-1 and TRADD bind differently to FAS-R and p55-R and

They also bind to each other. Further, both MORT-1 and TRADD bind effectively to RIP. Therefore, the interaction between these three proteins, MORT-1, RIP and TRADD, appears to be a significant component of the overall modulation of intracellular signaling mediated by these proteins. Interference of interactions between these three intracellular proteins will result in modification of the effects caused by these interactions. For example, inhibition of TRADD binding to MORT-1 may affect the interaction between FAS-R-p55 TNFR. Similarly, inhibition of RIP together with the above inhibition of TRADD binding to MORT-1 may further affect the interaction between FASR-p55 TNF-R.

Monoclonal antibodies directed against the death domain of p55-R, more specifically against the TRADD and RIP binding site, can also be used to inhibit or prevent the binding of these proteins and thus to modulate interactions between FAS-R and p55-R.

Recently, it has been found that in addition to the above-mentioned cytotoxic activities and their effects mediated by various receptors and their binding proteins including FAS-R, p55-R, TRADD, RIP, MACH, Mch4 and G, many of these receptors and their binding proteins are also involved modulating the activity of the nuclear transcription factor NF-κΒ, which is a key mediator of cell viability and survival, and which is responsible for controlling the expression of many immune and inflammatory response genes. For example, it has been found that TNF-α can induce two different types of signals in a cell, one inducing cell death and the other protecting the cell from inducing death by inducing gene expression via NF-κΒ (see Beg and Baltimore, 1996; Wang et al. , 1996; Van Antwerp et al., 1996). Similar double effects for FAS-R have also been described (see reference to this effect as reported by Van Antwerp et al., 1996). Therefore, there seems to be a delicate balance between cell death and cell survival after stimulation of different cell types of TNF-α and / or FAS-R ligands, where the ultimate effect depends on which intracellular pathway is more strongly stimulated, where one leads to cell death (usually apoptosis), and the other leads to cell survival through NF-kb activation.

Furthermore, the present inventors have recently evaluated the possible pathway by which members of the TNF / NGF receptor family activate NF-κB (see Malinin et al., 1997 and references cited therein; and related Israeli patent applications Nos. IL 117800 and IL 119133). Briefly, several members of the TGF / NGF receptor family appear to be able to activate NF-κB via a common adapter protein, TRAF2. A newly discovered protein kinase designated NIK (Malinin et al., 1997 and IL 117800 and IL 119133) is capable of binding to TRAF2 and stimulating NF-κΒ activity. Indeed, it has been shown (see Malinin et al. And IL applications) that expression in NIK kinase deficient mutant cells results in cells not being able to stimulate NF-κΒ in the normal endogenous manner, and also in blocking the induction of NF- κΒ TNF activity, either through FAS-R or TRADD, RIP and MOT-1 (which are adapter proteins that bind to these p55-R and / or AFS receptors). The p55-R, p75-R, FAS-R receptors and their adapter proteins MORT-1, TRADD, and RIP all bind to TRAF2, which by its ability to bind NIK clearly modulates NF-κB induction.

Of the above modulating proteins, which participate in the delicate balance between cell death and cell survival after stimulation with FAS-R and / or p55-R, the RIP protein appears to play an important role. RIP (see Stanger et al., 1995; and also Martini et al., 1997) contains a lethal domain in its C-terminal region, thanks to which it can induce cellular cytotoxicity in an independent manner or also through association of a lethal domain with lethal MORT domains. -1, p55-R, FAS-R, and TRADD. RIP contains, at its N-terminal region, a protein kinase domain and a middle domain that is likely to allow binding to TRAF2 and thus participate in NF-κB induction. Details of the characteristics and sequences (DNA and amino acid) of RIP are given in the above publications (especially Stanger et al., 1995), which are incorporated herein by reference.

TNF is also one of the cytokines involved in the initiation and modulation of host antiviral defense. Similarly, viruses have developed expression of genes whose proteins regulate cytokine activities, and these cytokine regulating viral proteins are believed to induce virus persistence in an animal host. One of the best studied examples of such proteins is E3-17.7K from group C of human adenovirus (Ad) types 2 and 5, which acts as a potent antagonist of TNF-mediated cytolysis.

In order to isolate the molecular components of the TNF signaling cascade that target E3-14.7K in viral infection, the human E3-14.7K binding protein was recently isolated by two-hybrid screening (FIP-2 according to Fourteen K Interacting Protein, Li, Y et al. , 1998). FIP-2 has been found to be non-toxic in itself and to reverse the protective effect of E3-14.7K on cytotoxicity induces overexpression of TNFR-I or RIP, without binding to any of the above proteins. FIP-2 has been found to have some homology to RAP-2, a protein of the present invention. However, the degree of overall similarity between RAP-2 and FIP-2 is very low, as seen from the overall assembly of the two amino acid sequences (Fig. 3). Homology is more significant in specific regions towards the C-terminus ♦ · · • · <

9 9 9 9 9 9 9 9 9 9 9 9 9 9 Proteins that culminate essentially in the identity of the 30 C-terminal amino acids. Interestingly, in addition to the above-mentioned C-terminal domain, the putative leucine finger motif in FIP-2 is largely retained in RAP-2 (except for substitution of Ile for Ala).

A similar sequence referred to as HYPL - a coding protein associated with Huntington's disease, which appears to be a remote homolog of RAP-2 - was recently deposited at GenBank under the name huntingtin interacting protein, HYPL (Accession No. AF049614). However, work describing the function of this protein has not been published.

A recent publication by Yamaoka, S: et al., 1998, describes the identification of a murine RAP-2 homolog. The murine homologue NEMO (essential modulator NF-κB) has been identified in the research of key molecules that regulate activation of NF-κΒ signaling. A complete cell variant of HTLV-1 Taxtransformed rat fibroblasts, designated 5R, was characterized and was non-reactive for all NF-κB activation stimuli tested (LPS, PMA, IL-1, TNF) and genetically complemented with this variant protein. Thus, a cDNA encoding the NEMO 48 kD protein was obtained. According to these data, this protein is absent from 5R cells, is part of the high molecular weight Ικ-B kinase complex and is required for its formation. In vitro, NEMO can homodimerize and interact directly with ΙΚΚβ.

Israeli Patent Application No. 120485 describes a RIP-associated protein, designated RAP, that specifically binds to RIP and inhibits NF-κB induction.

Israeli Patent Application No. 122758 discloses another RIP-associated protein, designated RAP-2, having the same or &lt;

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The RAP-2 of the present invention is also referred to as

303 or RAP-303 or RAT-303. It will hereinafter be referred to as RAP-2 in this application.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novel RAP-2 protein, including isoforms, analogs, fragments or derivatives thereof, that binds to a RIP protein (hereinafter referred to as RIP). RIP may directly or indirectly interact with intracellular mediators of inflammation, cellular cytotoxicity or cell death, such as p55-R and FAS-R, and their associated adapter or modulator proteins, such as MORT-1, TRADD, MACH, Mch4, G1 and others , and the novel proteins of the present invention may therefore, through binding to RIP, affect the intracellular signaling initiated by the binding of FAS ligands to their receptor and TNF to its receptor (p55-R), and such novel proteins of the present invention are modulators of cell-mediated effects of p55 -R and FAS-R. RIP may also interact with TRAF2, and thus may interact directly or indirectly with NIK, and as such, RIP acts as an inflammatory and cell survival modulator that includes NF-κB induction and hence the novel proteins of the present invention are RIP-related inflammatory and cell survival modulators. Similarly, through FAS-R, p55-R and their modulatory proteins MORT-1 and TRADD, which are capable of inducing NF-κΒ and cell survival either directly or indirectly by binding to RIP or by binding to TRAF2 to which RIP binds, The proteins of the present invention may also be mediators of cellular survival processes acting through common or related intracellular signaling pathways in which the aforementioned

proteins act to induce cell survival. Similarly, since p75-R binds to TRAF2 to which RIP binds, the proteins of the present invention can also be modulators of RIP-mediated p75-R mediated activity.

Another object of the present invention are antagonists (e.g., antibodies, peptides, organic compounds, or even some isoforms) of the above novel RAP-2 proteins, isoforms, analogs, fragments and derivatives thereof that can be used to inhibit signal transduction, or more specifically, inflammatory cellular cytotoxicity or processes involved in cell survival.

It is a further object of the invention to use novel RAP-2 proteins, isoforms, analogs, fragments and derivatives thereof for the isolation and characterization of other proteins or factors that may be involved in regulating receptor activity, for example, other proteins that can bind to RAP-2 proteins and which may affect their activity, and / or to isolate and identify other receptors involved in said signal transduction pathway to which these novel proteins, analogs, fragments and derivatives bind and therefore also affect their function.

The invention also includes RAP-2 binding proteins that can modulate / mediate the function of RAP-2.

Yet another object of the invention are inhibitors that can be introduced into cells where they can bind to or interact with RAP-2 and possible isoforms of RAP-2, wherein these inhibitors can inhibit RIP-related activity in cellular cytotoxicity processes and thus can, if if desired, they may enhance cell survival, or may inhibit RIP-related activity in cell survival processes, and so may if:

it is desirable to enhance cellular cytotoxicity.

It is a further object of the present invention to use the aforementioned RAP-2 proteins, isoforms and analogs, fragments and derivatives thereof as antigens for the preparation of polyclonal and / or monoclonal antibodies to these substances. The antibodies can then be used, for example, to purify novel proteins from various sources, such as cell extracts or transformed cell lines.

Furthermore, these antibodies can be used for diagnostic purposes, for example, to identify diseases associated with abnormal function of cellular processes mediated by p55-R, FAS-R, or other related receptors.

A further object of the present invention are pharmaceutical compositions comprising novel RAP-2 proteins, isoforms and analogs, fragments and derivatives thereof, as well as pharmaceutical compositions comprising the above antibodies or other antagonists.

In the present invention, a novel RAP-2 protein was isolated. RAP-2 can interact with or bind to RIP and therefore is a modulator or mediator of intracellular activity of RIP. RIP is involved in modulating or mediating intracellular signal transduction, for example, signaling pathways associated with cellular cytotoxicity or cell death, in which RIP has cytotoxic activity by itself or in conjunction - directly or indirectly - with other proteins associated with cell death, such as MORT -1, TRADD, MACH, Mch4, GI, p55-R and FAS-R with which RIP can associate or bind directly or indirectly through the lethal domain motif / module present in RIP and all above said proteins; another signaling pathway is the inflammatory pathway, cellular survival or viability in which it may have an inflammatory pathway. RIP activation role, either directly or indirectly through a kinase motif or domain present in RIP and the ability of RIP to bind to TRAF2, which may bind to NIK, which ultimately directly participates in NK-κΒ activation, which is central to inflammation and survival cells. In addition, p55-R may also interact with TRADD and TRAF2 (via TRADD) and is also believed to be involved in NK-κΒ activation and thus in the cell survival pathway, and therefore RIP that binds to or interacts with FAS- R, TRADD and p55-K (via TARDD), as well as with TRAF2, also participate in modulating inflammation and cellular survival by these proteins. Therefore, the RIP modulator or mediator of these signaling pathways and the like is the new RAP-2 of the present invention is the RIP modulator or mediator of these signaling pathways.

RAP-2 was isolated and cloned using a yeast two-hybrid system, sequenced and characterized, and, as further detailed below, RAP-2 appears to be a highly specific RIP binding protein and therefore a specific modulator / mediator of RIP. RAP-2 does not bind to TRADD, MORT-1, p55R, p75-R, and MACH. Furthermore, it appears that RAP-2 does not contain the characteristic death domain module, consistent with the finding that RAP-2 does not induce cytotoxicity per se.

As used herein, the term RIP activity refers to its activity in modulating / mediating inflammation and cell death / survival. These activities are set forth hereinabove and hereinafter, as well as in all of the above-mentioned publications and patent applications, the contents of which are hereby incorporated by reference in their entirety. Similarly, as used herein, the term RAP-2 refers to modulating / mediating RIP activity through specific binding to RIP, wherein

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4444 4 modulating / mediating RIP activity RAP-2 includes modulating / mediating signal transduction in inflammation, cell death, and cell survival in which RAP-2 is directly or indirectly involved and in this sense RAP-2 can be considered an indirect modulator / mediator of all of the aforementioned proteins, and perhaps many others, involved in mediating signal transduction in inflammation, cell death and cell survival and to which RIP binds, or interacts with RIP directly or indirectly.

The invention also discloses two novel RAP-2 binding proteins that have been identified by two-hybrid screening using the complete RAP-2 protein sequence as a capture molecule.

Using the complete RAP-2 protein as capture molecule in a two-hybrid assay, a novel RAP-2 interacting protein identified in the present invention as clone # 10 (or protein encoded by clone # 10 or RAT binding protein # 10 or RBP-10) was identified. . The obtained cDNA sequence was further expanded using conventional sequencing methods towards the 5 'end to re-create a partial open reading frame of the protein but lacking the start codon.

The two-hybrid binding repertoire assay of clone # 10 showed that this protein not only binds to RAP-2, but also has a fairly strong affinity for TRAF2. However, clone # 10 does not bind to RIP, TRADD, MORT1, MACH, TNFR-1, TIP60, and NIK, as well as several control proteins (e.g., lamin and cyclin D). However, it cannot be excluded that the binding of clone # 10 to NIK can be detected in mammalian cells due to the peculiarities of the behavior of NIK in yeast. Clone # 10 has been shown to bind to RAP-2 in the C-terminal 200 amino acids of RAP-2, i.

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949 in a region not necessarily associated with RIP, TIP60, NIK, and ββ binding. This sequence allows, inaccurately, several GenBank search rounds to identify clones # 10 homologs. The only protein showing a significant degree of similarity to the protein encoded by clone # 10 was F40F12.5 - a hypothetical C. elegans molecule whose physiological role is unknown.

Interestingly, F40F12.5 shows some similarities to several members of the highly conserved ubiquitin-protease family. These enzymes neutralize the destructive effects of the ubiquitination apparatus, which is involved in most of the events associated with protein degradation in the cell. While ubiquitin ligases are responsible for binding the polyubiquitin chain to a protein to be degraded, ubiquitin proteases prevent efficient branching of the growing chain. However, the predicted function of F40F12.5 based on similarity to the above-mentioned ubiquitin-driven proteases is questionable and it has not yet been established whether this protein has any enzymatic activity with respect to ubiquitin polymers. In addition, several facts make such coincidence quite unlikely:

(a) residues believed to form the core of the catalytic region in any ubiquitin-protease subclass are not conserved in either F40F12.5 or clone # 10;

(b) with the exception of their catalytic sites, enzymes from the ubiquitin-driven protease family from different species (from bacteria and humans) show essentially no sequence similarity, whereas F40F12.5 and clone # 10 show some degree of homology.

RAP-2 appears to be a specific binding protein for RIP and therefore a modulator / mediator of the intracellular activity of RIP. RAP-2 ·

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The binding proteins may, through their ability to bind to RAP-2, indirectly affect RIP and are therefore modulators / mediators of the intracellular activity of RIP.

Therefore, RAP-2 clearly plays a role in modulating / mediating signal transduction in inflammation, cell survival, and / or cell death in which RIP is directly or indirectly involved, particularly those related to cytotoxicity and inflammation caused or induced by various stimuli, including those transmitted via receptors of the TGF / NGF receptor family and perhaps over other receptors (for a scheme of involvement of RIP - and thus RAP-2 - in these intracellular events, see Figure 1 in Malinin et al., 1997).

RAP-2 can also serve as an inhibitor of cytotoxicity and inflammation by being present as part of a complex of other proteins, such as RIP and proteins bound to RIP, and as such may affect the cytotoxic and inflammatory effects of these other proteins (e.g. p55-R, FAS (R, MACH, Mch4, G1, and MORT), resulting in inhibition of their cytotoxic activity or inflammatory activity.

RAP-2 can also serve as an enhancer or enhancer of cytotoxicity and inflammation by enhancing the activity of other proteins, such as RIP and other proteins bound to RIP, as mentioned above, by assisting the binding of these proteins to RIP, enhancing the cytotoxic activity of various proteins or enhances their inflammatory effects.

Similarly, in an analogous manner, RAP-2 may also serve as an inhibitor or enhancer of cell survival as described above by affecting RIP in this pathway.

Accordingly, the present invention provides a DNA sequence

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coding for a protein associated with RIP (RAP-2, its isoforms, analogs or fragments thereof, which are capable of binding to RIP and affecting or mediating intracellular activity of RIP, wherein said intracellular activity is RIP-2. the activity affects / mediates inflammation and / or cell death and / or survival.

More specifically, the present invention provides a DNA sequence selected from the group consisting of:

(a) a cDNA sequence derived from the coding region of the native RAP-2 protein;

(b) a DNA sequence capable of hybridizing to (a) under moderately stringent conditions that encodes a biologically active RAP-2 protein; and (c) DNA sequences that are degenerate due to the degeneracy of the genetic code relative to the DNA sequences defined in (a) and (b) and which encode a biologically active RAP2 protein.

Another specific embodiment of the above DNA sequence of the present invention is a DNA sequence comprising at least a portion of a sequence encoding at least one RAP-2 protein isoform. Another embodiment of the above DNA sequence is the sequence encoding the RAP-2 protein as shown in Figure 1. Yet another embodiment is the DNA sequence shown in Figure 2.

The present invention provides RAP-2 proteins and analogs, fragments or derivatives thereof encoded by any of the above sequences of the present invention, wherein said proteins, analogs, fragments and derivatives are capable of binding to RIP and affecting / mediating its biological activity in intracellular signal transduction pathways. to cell death / survival.

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A specific embodiment of the invention is the RAP-2 protein, analogs, fragments and derivatives thereof. The sequence of the RAP-2 protein as derived from the DNA sequences of Figures 1 and 2 is shown in Figure 3.

Another embodiment is any isoform of RAP-2 protein, analogs, fragments and derivatives thereof.

The present invention also includes replicable expression vehicles comprising the above DNA, wherein the replicable expression vehicles are capable of being expressed in suitable eukaryotic or prokaryotic host cells; transformed eukaryotic or prokaryotic host cells comprising such replicable expression vehicles; and a method for producing the RAP-2 protein or analogs, fragments or derivatives thereof by culturing such transformed host cells under conditions suitable for expression of said protein, analogs, fragments or derivatives thereof, making post-translational modifications of said protein necessary to obtain said protein and extracting said protein, analogs, fragments or derivatives thereof from the culture medium of said transformed cells or from cell extracts of said transformed cells. The above definitions include all isoforms of the RAP-2 protein.

In another aspect, the present invention also provides antibodies or active derivatives or fragments thereof specific for the RAP-2 protein and analogs, fragments and derivatives thereof of the present invention.

In yet another aspect, the invention provides various uses of the above DNA sequences, including:

(i) method for affecting intracellular signal transmission

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4444 9 ·· on inflammation, death and / or survival of cells which is affected or mediated by RIP, which comprises treating said cells with one or more RAP-2 proteins, isoforms, analogs, fragments or derivatives thereof that can bind to RIP, wherein said treatment comprises introducing said proteins, isoforms, analogs, fragments or derivatives thereof in a form suitable for their intracellular transfer into said cells, or inserting a DNA sequence encoding said proteins, isoforms, analogs, fragments or derivatives thereof in the form of a suitable vector carrying said sequences to said cells, wherein said vectors can mediate insertion of said sequence into said cells such that said sequence is expressed in said cells;

(ii) a method for affecting intracellular signal transduction in inflammation, death, and / or cell survival, which is mediated by TNF family cell ligands via a RIP protein, performed according to the method of the above (i), wherein said treatment comprises inserting said RAP -2 a protein, isoforms, analogs, fragments or derivatives thereof in a form suitable for their intracellular transfer into said cells, or insertion of a DNA sequence encoding said G1 protein, isoforms, analogs, fragments or derivatives thereof, in the form of a suitable vector carrying said sequences cells, wherein said vectors can mediate insertion of said sequence into said cells such that said sequence is expressed in said cells;

(iii) the method of method (ii), wherein said treating cells is performed by transfecting said cells with a recombinant animal viral vector, comprising the steps of:

(a) construction of recombinant animal viral vector • 4 • 4 4

4 · · 4 4

4 carrying a sequence encoding a viral surface protein (ligand) capable of binding to a specific cell surface receptor on the surface of cells carrying FAS-R or p55-R and a second sequence encoding a protein selected from RAP-2 protein, isoforms, analogs, fragments and derivatives which, upon expression in said cells, are capable of influencing / mediating signal transduction pathways in inflammation, death and / or cell survival; and (b) infecting said cells with said vector of step (a);

(iv) a method for affecting intracellular signal transduction in inflammation, death and / or cell survival, which is mediated by TNF family cell ligands via a RIP protein, which comprises treating cells with antibodies or active fragments or derivatives thereof of the present invention, wherein said treatment is performed by applying a suitable composition comprising said antibodies, active fragments or derivatives thereof to said cells, said composition being ready for extracellular administration when at least a portion of the RAP-2 protein is present on the extracellular surface, and wherein said composition is ready for intracellular administration, when said RAP-2 proteins are located completely intracellularly;

(v) a method for affecting intracellular signal transduction in inflammation, death and / or cell survival, which is mediated by TNF family cell ligands via a RIP protein, comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence to at least a portion of the RAP-2 protein sequence according to the present invention, wherein said oligonucleotide sequence is capable of blocking the expression of the RAP-2 protein;

• ·

·. (Vi) a method according to method (ii) for treating cancer cells or cells infected with HIV or other cells afflicted with the disease, comprising:

(a) constructing a recombinant animal viral vector carrying a sequence encoding a viral surface protein capable of binding to a specific cell surface receptor on the surface of tumor cells or to a specific surface receptor on the surface of HIV infected cells or to a receptor on other cells affected by the disease; selected from the RAP-2 protein, its isoforms, analogs, fragments and derivatives which, upon expression in said tumor cells, HIV-infected cells or other cells afflicted with the disease, is capable of killing said cells by acting on the RIP protein; and (b) infecting said cells with said vector of step (a);

(vii) a method for affecting intracellular signal transduction at cell death and / or survival, which is mediated by TNF family cell ligands via a RIP protein, comprising the use of a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding the RAP-2 protein of the present invention; which is introduced into said cells in a form allowing expression of said ribozyme sequence in said cells and wherein said ribozyme sequence upon expression in said cells interacts with said cell mRNA sequence and cleaves said mRNA sequence resulting in inhibition of expression of said RAP-2 protein in said cells cells;

(viii) a method selected from the above methods of the present invention, wherein said sequence encoding the RAP-2 protein comprises at least one of the isoforms, analogs, fragments and derivatives of the RAP-2 of the present invention that can bind to RIP;

(ix) a method for isolating and identifying proteins of the present invention which are capable of binding to a RIP protein, comprising applying a yeast two-hybrid method, wherein the sequence encoding said RIP protein is carried on a single hybrid vector and sequence from a cDNA library or genomic library The DNA is carried on a second hybrid vector, which vectors are then used to transform yeast host cells and the positive transformed cells are isolated, and then extracting said second hybrid vector to obtain a sequence encoding a protein that binds to said RIP protein.

(x) the method of any one of methods (i) - (x), wherein said RAP-2 protein is any RAP-2 isoform or any analog, fragment or derivative thereof;

(xi) a method according to any one of methods (i) - (x), wherein said RAP-2 protein or any of its isoforms or any analog, fragment or derivative thereof is involved in influencing cell events mediated or modulated by any other mediator or inducer, to which directly or indirectly binds said RAP-2 protein, isoform, analog, fragment or derivative thereof.

The present invention also provides a pharmaceutical composition for affecting signal transduction pathways in inflammation, cell death and / or cell survival mediated by the effect of TNF acting on the RIP protein or any other mediators or inducers, said composition comprising as an active ingredient one of the following:

(i) the RAP-2 protein of the present invention and its

9 9 · * * * * * * * * *

Φ * ♦

Biologically active fragments, analogs, derivatives or mixtures thereof;

(ii) a recombinant animal viral vector encoding a protein capable of binding to a cell surface receptor and encoding the RAP-2 protein or biologically active fragments or analogs thereof of the present invention;

(iii) an oligonucleotide sequence encoding an antisense sequence to the RAP-2 protein sequence of the present invention, wherein said oligonucleotide sequence may be the second sequence of the recombinant animal viral vector of (ii).

The present invention also provides:

I. a method for affecting inflammatory, cell death and / or survival signal transduction pathways that are modulated / mediated by RIP or any other mediators or inducers or any other inducers or inhibitors of NF-κΒ in cells, said method comprising treating said cells with a method of said methods (i) - (x) using RAP-2 proteins, isoforms, analogs, fragments or derivatives thereof or using sequences encoding RAP-2 proteins, isoforms, analogs or fragments thereof, wherein said treatment results in activation or inhibition said events mediated by RIP and thereby also events mediated by FAS-R or p55-R, or another said mediator or inducer or another inducer or inhibitor of NF-κB;

II. the method of the above, wherein said RAP-2 protein, analog, fragment or derivative thereof is that portion of the RAP-2 protein that specifically participates in binding to RIP or to said other mediator or inducer or to another inducer or mediator NF-κΒ, or wherein said sequence encoding said RAP2 protein encodes that portion of the RAP-2 protein that specifically

• participates in the binding to RIP or to another mediator or inducer, or to another inducer; or NF-κB mediator;

III. the method set forth above, wherein said RAP-2 protein is any of the RAP-2 isoforms, wherein said isoform is capable of enhancing the effects associated with RIP;

IV. the method set forth above, wherein said RAP-2 protein is any of the RAP-2 isoforms, wherein said isoform is capable of inhibiting the effects associated with RIP or effects associated with another mediator or inducer and therefore also inhibiting effects associated with FAS-R or p55 -R or effects of other cytotoxic mediators or inducers;

V. the above method, wherein said RAP-2 protein, isoform, analog, fragment or derivative thereof is capable of enhancing or inhibiting RIP-related effects on signal transduction pathways associated with inflammation or cell survival through direct or indirect inhibition of NK-κB or direct or indirect activation of JNK or p38 kinase.

Isolation of RAP-2 proteins, their identification and characterization can be performed using any standard techniques for protein isolation and identification, for example using the yeast two-hybrid method, affinity chromatography methods, and any well known methods suitable for this purpose.

In yet another aspect of the invention, the RAP-2 protein itself or an isoform, fragment or derivative thereof is used as a capture agent in a yeast two-hybrid assay to obtain proteins binding to the protein.

• · · · · · ·

Proteins that bind to RAP-2, its isoforms, fragments or derivatives are also part of the present invention.

Other aspects and embodiments of the present invention will be apparent from the detailed description of the invention.

It is to be understood that the terms affecting / mediating the effects of RIP, FAS-ligands or TNF on cells and any other such affecting / mediating used in the present invention include affecting both in vitro and in vivo as well as inhibiting or enhancing / enhancing the effects.

Description of the figures in the attached drawings

Giant. 1 (A, B) (SEQ ID NO; 1) shows the nucleotide sequence

RAP-2, in which the start and stop codons are underlined. The arrows indicate the start of a 1.5 kb clone obtained in a two-hybrid screen;

Giant. 2 (A, B) (SEQ ID NO: 2) shows the nucleotide sequence of clone # 41072 (see Example 1), in which start and stop codons are underlined.

Giant. 3A (/ 1, / 2) show the derived amino acid sequence of human (20.4 complete and Human shrt) and mouse (NEMO complete and Mouse part) spliced variants of RAP-2 and B (/ 1, / 2) show published FIP- 2 assigned using a software package available from BCM Search Launcher (Baylor College of Medicine, Houston, TX). Homologous amino acids are framed, identical amino acids are in gray boxes. The asterisks in (B) indicate the putative leucine finger-like motifs (LZ) in FIP-2.

• ·

Giant. 4 describes the molecular characterization of RAP-2. Northern hybridization of human MTN Blot I (Clontech) with the RAP-2 DNA fragment is analyzed. VB is analyzed for RAP-2 binding to RIP as described in more detail in Example 3. VC is detected by the NIK-RAP-2 interaction as in (B), except that anti-FLAG antibodies were used for western blotting and then followed by immunoprecipitation with anti-His6. The arrows indicate the positions of the immunoprecipitated proteins.

Giant. 5 is a graphical representation of the massive inhibition of NFkB and c-Jun activation caused by various stimuli achieved by ectopic expression of RAP-2 as described in Example 4. HEK-293T cells were transiently transfected with a reporter plasmid (HIVLTR-Luc or CMV-Luc for NF-κB (A) and GAL4-Luc for c-Jun (Β) activation assays) and an expression vector for said inducer and cell with empty vehicle (pcDNA3 - indicated separately) or plasmid encoding complete RAP-2 (pcRAP-2 - marked plus in the picture). Activation of the luciferase activity of the reporter gene is expressed in relative luciferase units (RLU).

Giant. 6 shows that RAP-2 exhibits similar inhibitory behavior towards NF-κB and c-Jun at different concentrations. TRAF2 was transiently expressed in HEK-293T cells along with various amounts of either the pcRAP-2 (coding) or pcRAP-2-a / s (antisense) construct. PHIVLTR-Luc and pGAL4-Luc reporter plasmids were used to evaluate NF-κB (A) and c-Jun (B) activation. The Luciferase assay was performed as described for Figure 5 in Example 4.

Giant. 7 shows that RAP-2 potently potentiates signal-induced c-Jun phosphorylation without interfering with JNK1 / 2 activation.

(A) total cell lysates of HEK-293T cells transfected with said expression constructs together with either the ppcDNA3 carrier indicated by (-) in the figure or with pcRAP2 indicated by (+) in the figure were analyzed by Western blot with anti-phospho-Jun antibodies as described in Example 5. The control membrane indicated on the lower panel was probed using antibodies against total c-Jun (NEB);

(B) Activated JNK1 / 2 from HEK-293T cells transfected with either pcDNA3 or pcRAP-2, treated with hrTNFα for an extended period of time were analyzed by Western blotting of total lysates with anti-phospho-JNK antibodies as detailed in Example 5.

(C) HEK-293T cells were co-transfected with the empty vector, pcRAP-2 and pcRIP in various combinations, together with the HAJNK1-expression plasmid. Then, JNK1 was immunoprecipitated using the N-terminal HA-terminal and its ability to phosphorylate GST-Jun produced in bacteria was determined in an in vitro kinase assay. Reaction products were analyzed by SDS-PAGE. GST-Jun is indicated by an arrow.

Giant. 8 shows that RAP-2 does not compete with NF-κΒ and AP-1 for binding

DNA. HEK-293T were transfected with the indicated proteins alone (-) or together with pcRAP-2 (+). Nuclear extracts prepared from the cells were co-incubated with ³² P-labeled oligonucleotides containing the classical recognition sequences for AP-1 (A) or NF-κΒ (B). Reaction products were analyzed by non-denaturing PAGE.

Giant. 9 shows that RAP-2 affects basal NF-κΒ levels

HEK-293T and HeLa cells transiently transfected with a variable amount of either RAP-2 (coding) or RAP-2 antisense (a / s). All operations were performed as described for FIG.

Example 4.

Giant. 10 (A, B) (SEQ ID NO: 3) shows the partial nucleotide sequence of clone # 10.

Giant. 11 shows the functional properties of serial RAP-2 deletions.

A is a schematic representation of sequential C-terminal deletions in RAP-2. All deletions retained the intact N-terminus of RAP-2, while the C-terminus is indicated by arrows. The binding region for RIP, NIK, β and TIP 60 is underlined. The three hatched boxes correspond to the supposed leucine finger motifs. B shows the effect of overexpression of the deletion constructs described in A on NF-κB activation in HEK-293T cells by RelA, TRAF2 TNF and NIK using the HIV-LTR luciferase reporter plasmid for NF-κΒ. Activation of the luciferase activity of the reporter gene is expressed in relative luciferase units (RLU).

Giant. 12 shows the mapping of RAP2 functional and binding regions.

(A) Various deletion variants of RAP-2 were tested for their ability to bind to said proteins in transfected yeast (odd bars) and in mammalian HEK-293T cells (even bars). The two rightmost bars show the ability of the same deletion variants transfected in high amounts as described in Example 9 into HEK-293T cells to inhibit NF-κB activation and potentiate hyperphosphorylation (c-Jun) in response to TNF-α administration. The thickness of the crosses is proportional to the intensity of the effect. Asterisks indicate that the observed effects of labeled constructs on Rel-A stimulation are different (see Figure 11B).

(B) Summary graph representing the localization of the binding (upper) and functional (lower) regions of RAP-2, as was

4 * 4 * ·· ·· from the deletion analysis shown in Figure (A), relative to the protein skeleton. The hatched parts are the possible localization of the boundaries of the respective minimal regions.

Giant. 13 shows that ser-148 in RAP-2 is required for its ability to induce c-Jun hyperphosphorylation to ser-63.

A Western blot is shown in which wt is a wild type, S148A indicates that the ser at position 148 has been replaced by Ala and the vector is an empty control vector.

The present invention relates, in one aspect, to novel RAP-2 proteins that can bind to the RIP protein and thus affect or mediate the intracellular activity of RIP, particularly where RIP participates in signal transduction pathways associated with inflammation, death and / or survival cells. Thus, RAP-2 may inhibit RIP activity in the cell death / inflammation / survival pathway, RAP-2 may enhance RIP activity in the cell death / inflammation / survival pathway, or it may enhance RIP activity in one of these pathways and inhibit it in another of these of tracks.

More specifically, the present invention provides a novel RAP-2 protein. RAP-2 has been sequenced and characterized, and RAP-2 has been found to be an RIP-binding protein having high specificity for RIP but does not bind to many proteins known to be involved in signal transduction pathways leading to inflammation, cell death or cell survival. RAP-2 also clearly has no domain common to proteins that are active in one of these pathways, i.e., RAP-2 has no motif or death domain module, no kinase motif or domain, and no protease motif or domain. The sequence of RAP-2 is also a unique sequence, as determined by comparison to sequences from many databases, including GenBank, Human Genome Level 1, and the database databases. How

4

4

4 is discussed in more detail below (and also in all cited references and patent applications), involved in RIP intracellular signal transduction pathways leading to inflammation, cell death, and cell survival. Therefore, regulation or control of RIP activity may regulate some or all of these pathways where these pathways are triggered, for example, by binding of TNF or FAS-ligands to its receptors (for TNF at p55-R). RIP may be of key importance in determining which pathway is activated to a greater extent and this is due to its ability to bind to many cytotoxic proteins having deadly domains, as well as to many proteins with kinase activity. Therefore, proteins such as the RAP-2 protein of the present invention, which can specifically bind to RIP, may be of value in influencing RIP activity and thus influencing the extent of induction of one pathway relative to other pathways. Therefore, the RAP-2 protein of the present invention is an important modulator or mediator of intracellular signal transduction.

In addition to the full length RAP-2 protein of the present invention, a shorter cDNA was cloned, which was found to be composed of sequence blocks derived from several remote regions of the full length cDNA, and which clearly originates from alternative splicing of the same gene. The mouse counterpart of human RAP-2 was identified in a similar analysis of the mouse EST file. Partial murine cDNA was found to be substantially identical to the human counterpart in the coding region.

The physiological significance of the RIP-RAP-2 interaction was further confirmed in transfected HEK-293T and HeLa cells. However, the formation of such complexes did not result in the enzymatic activity of RIP, as evidenced by the fact that overexpressed RIP does not phosphorylate RAP-2.

Transfection experiments on mammalian HEK-293T cells also resulted in 9 9 9 9 9

9 9 9 for stable formation of the RAP-2-NIK complex.

RAP-2 appears to be an essential element in NF-κB and c-Jun signal transduction pathways as it binds to NIK, β and TIP60 (histoacetyltransferase) and modulates NF-κΒ and c-Jun-dependent transcription. Indeed, enhanced ectopic expression of RAP-2 leads to inhibition of the NF-κΒ response, while its depletion from cells by an antisense construct - leads to enhanced transactivation of NF-κB and c-Jun.

RAP-2 has also been found to potentiate cJun hyperphosphorylation that is not mediated by JNK activity. RAP-2 does not inhibit c-Jun and RelA binding to DNA. RAP-2 binding and functional domains were identified by sequence deletion analyzes. These studies have shown that the binding region for RIP, NIK and TIP60 overlap and is found in amino acids 95-264 of RAP-2. However, the subsequent functional effects mediated by RAP-2 were localized to the N-terminal domain of the protein consisting of amino acids 1-264.

For the above reasons, RAP-2 appears to be an essential element in the signal attenuation pathway of the stress response: ectopic expression of the coding construct inhibits the response, while expression of the antisense construct enhances the response. RAP-2 is also known in the laboratory of the present inventors as RAT (RIP attenuator) and can therefore also be referred to as RAT and / or RAT-303 and / or clone 303.

The existence of multiple splice variants suggests that - at least in part - the pure effect of RAP-2 under the given conditions is likely to be dependent on the presence of some sequence blocks required for protein binding / targeting / translocation / modification in the predominant isoform. For example, if RAP-2 and TIP60 binding allow nuclear localization of RAP-2, then RAP-2 variants with excised nuclear localization signals (NLS) will be defective or, conversely, active in inhibition. NF-KB / AP-1. Sequence analysis showed that RAP-2 contains several positively charged amino acid groupings (E, K and R) characteristic of most known NLS.

The binding of RAP-2 to RIP was mapped to the RAP-2 region of the protein that begins between amino acids 117-218 and ends with amino acid 264. The RIP binding domain in RAP-2 does not overlap with either the β-beta or NIK binding site.

Binding to TIP60, which is a member of nuclear proteins referred to as histone acetyltransferases, was mapped to the region between amino acids 95-264. The region involved in homodimerization was located between amino acids 217-264.

The data obtained suggest that all functional effects of RAP-2 (specifically NF.kB inhibition and c-Jun hyperphosphorylation induction) localize to the same region.

The protein encoded by clone # 10 binds in a region beginning between amino acids 218-309 and ending at amino acid 416, and therefore its binding site may contain overlapping regions with binding sites for RIP, NIK, β, and TIP60.

Further, it is possible that a region sufficient to efficiently influence signaling mediated by all other inducers is localized to the N-terminal segment of the protein.

The region formed by amino acids 95-416 has an effect, although significantly weaker compared to the effect caused by the full-length protein, which may therefore be due to enhanced aggregation of endogenous RAP-2.

In addition, except for RelA, all the effects induced in our experiments can be mediated by only about 100 N-terminal amino acids of RAP-2. Indeed, even a fragment containing amino acids 1-102 mediates distinct effects, although weaker.

On the other hand, the suppression of RelA-mediated effects requires the use of a significantly longer portion of RAP-2. So far, we have located the boundaries of this region between amino acids 1-264, which clearly contain a region between amino acids 157-264 with certain specific RelA binding properties.

From the above observations, it seems that:

(a) with the exception of RelA, binding of RAP-2 to RIP, clone # 10 and most likely NIK and TIP60 is not required for protein function as an inhibitor of NF-κB-induced overexpression;

(b) the effect of RAP-2 on overexpression induced RelA activation is mediated, at least in part, by another binding event. Essentially all of the above proteins may contribute to said activity, as is evident from the experiments carried out to date.

Because of the unique ability of FAS-R and TNF receptors to cause cell death, as well as the ability of the TNF receptor to trigger other tissue damaging activities, impairment of the function of these receptors can be highly detrimental to the body. In addition, it has been shown that both excessive and deficient function of these receptors contribute to the pathological manifestations of various diseases. The identification of molecules involved in signal transduction from these receptors and the discovery of ways to affect the function of these molecules create opportunities for new therapeutic approaches to these diseases. In view of the predicted significant role of RIP in FAS-R and p55-R toxicity, and therefore, the predicted significant regulatory role of RAP-2 in FAS-R and TNF by influencing RIP seems to be of significant importance for the development of new drugs that could block cytotoxic RIP functions, perhaps by blocking the binding of RAP-2 to RIP or otherwise inhibiting the interaction between RAP-2 and RIP in these conditions in which RAP-2 potentiates RIP-mediated cytotoxicity (as noted above, RIP is cytotoxic both on its own and in association with other proteins containing regions of the death domains).

Similarly, it is also known (see above) that FAS-R and p55-R are involved in NF-κB activation and thus cell survival. Therefore, if killing of cells, such as tumor cells, HIV-infected cells and the like is desired, it is desirable to potentiate the cytotoxic effects of FAS-R and p55-R (and their associated proteins such as MORT-1, MACH, Mch4, Gl, TRADD ), while inhibiting their NF-kb inducing activity. Therefore, if the interaction or binding of RAP-2 to RIP leads to an enhancement of the possible role of RIP in enhancing NF-κB induction (probably via TRAF2 and probably via the kinase domain and / or the middle domain of RIP), then blocking this interaction between RAP-2 and RIP to inhibit, or at least prevent enhancement, activation of NF-κB and thus shift the balance of the effects induced by TNF or FAS-ligands to the side of cytotoxicity, ultimately causing cell death.

Similarly, in the opposite situation (relative to the situation above), in which the binding of RAP-2 to RIP causes inhibition of FAS-R and p55-R pro-inflammatory or cytotoxic effects and it is desirable to block these cytotoxic effects (e.g. inflammation, various autoimmune diseases and the like in which higher cell survival is desired), it is important to design drugs that will enhance the interaction between RAP-2 and RIP and thereby enhance overall cell death inhibition and shift the balance in favor of cell survival. It is also clear from the above that when the interaction of RAP-2 with RIP causes inhibition of RIP function in enhancing NF-κB activation and cell survival is desirable, it is necessary to block this interaction between RAP-2 and RIP, thereby enhancing RIP activity in enhancing NF-κB activation.

It follows from the above description that RIP plays a key role in the balance between inducing or mediating signaling pathways leading to inflammation, cell death or cell survival, and because RAP-2 plays an equally important role as it is a RIP modulator. Influencing the interaction / binding between RAP-2-RIP using various drugs as discussed above and below could allow for a shift in intracellular signaling pathways from cell death to cell survival and vice versa, as appropriate.

The present invention also relates to a DNA sequence encoding a RAP2 protein and RAP-2 proteins encoded by a DNA sequence.

Further, the present invention relates to DNA sequences encoding biologically active analogs, fragments and derivatives of the RAP-2 protein and analogs, fragments and derivatives encoded by said sequences. Preparation of such analogs, fragments and derivatives is accomplished by standard procedures (see Sambrook et al., 1989) in which one or more codons can be deleted, added, or substituted with other codons in DNA sequences encoding the RAP-2 protein to produce analogs containing at least one amino acid change relative to the native protein.

* »· · ·» »• • • •

Of the above DNA sequences of the present invention that encode a RAP-2 protein, isoform, analog, derivative or fragment thereof, the invention also includes, in one embodiment, a DNA sequence capable of hybridizing to a cDNA sequence derived from the coding region of the native RAP-2 protein. wherein the hybridization is performed under moderately stringent conditions, and wherein the hybridizable DNA sequence encodes a biologically active RAP-2 protein. These hybridizable DNA sequences therefore include DNA sequences that have relatively high homology to the natural RAP-2 cDNA sequence and as such represent sequences similar to the RAP-2 sequence, which may be, for example, natural sequences encoding different RAP-2 isoforms, natural sequences encoding proteins belonging to the group of sequences similar to RAP-2 sequences encoding proteins having RAP-2 activities. Further, these sequences may include, for example, unnatural, synthetic sequences that are similar to the natural RAP-2 cDNA sequences, but which contain many desirable modifications. Such synthetic sequences therefore include all possible sequences encoding RAP-2 analogs, fragments and derivatives, all of which have RAP-2 activities.

Standard procedures for screening and isolating natural DNA or RNA samples from various tissues using native RAP-2 cDNA or a portion thereof as probes can be used to obtain various of the above RAP-2 sequences similar to those described above (see, for example, standard procedures in Sambrook et al. al., 1989).

Similarly, a variety of standard procedures can be used to prepare the various RAP-2-like synthetic sequences coding for RAP-2 analogs, fragments, or derivatives described above, as described below for preparing such analogs, fragments, and derivatives.

4 4 «« * 4 4 · · · · 4 4 ·

• 445 4 «

The term polypeptide or protein substantially corresponding to RAP-2 protein, but also polypeptides or proteins that are analogs of RAP-2.

Analogues substantially corresponding to the RAP-2 protein are those polypeptides in which one or more amino acids of the amino acid sequence of the RAP-2 protein have been replaced by another amino acid, deleted and / or inserted, provided that the resulting protein exhibits substantially the same or higher biological activity as the RAP-2 protein to which they correspond.

To ensure that the proteins essentially correspond to the RAP-2 protein, the changes in the sequence of the RAP-2 proteins, such as its isoforms, are relatively small. Although the number of changes may be greater than 10, preferably no more than 10 changes are present, more preferably no more than 5 changes, and most preferably no more than 3 such changes. Although any technique can be used to discover potential biologically active proteins substantially corresponding to RAP-2 proteins, one such technique is a conventional mutagenesis technique performed on DNA encoding a protein that results in several modifications. Proteins expressed by such clones can then be examined for their ability to bind to RIP and for the ability to influence RIP activity in affecting / mediating the intracellular signal transduction pathways mentioned above.

Conservative changes are those that are not expected to alter the activity of the protein and are usually tested first because they are not expected to significantly alter the size, charge, or configuration of the protein and thus its biological properties.

4 · 4 · · * * * * «« «« 9 9 «9«

449449 4 49 49 4

9 4 4 4 9 4 5

4994 4,949,449 44 * 9

Conservative substitutions of RAP-2 proteins include analogs in which at least one amino acid residue of the polypeptide has been conservatively replaced by another amino acid residue. Such substitutions are preferably made according to the following list in Table IA, where such substitutions can be determined by routine experimentation to obtain modified structural and functional properties of the synthesized polypeptide molecule while maintaining the biological activity characteristic of the RAP-2 protein.

Table IA

The original rest

Ala

Arg

Asn

Asp

Cys

Gin

Glu

Gly

His

Ile

Leu

Lys

Met

Phe

Ser

Thr

Trp

Tyr

Wall

Example of Gly substitution; Ser Lys

Gin; His

Glu

Ser

Asn

Asp

Ala; Pro Asn; Gin Leu; Val Ile; Val Arg; Gin; Glu Leu; Tyr; Ile Met; Leu; Tyr Thr

Ser

Tyr

Trp; Phe Ile; Leu

Alternatively, another group of substitutions in the RAP-2 protein are those in which at least one amino acid residue in the polypeptide is deleted and another amino acid residue according to Table IB is inserted in its place. The types of substitutions that can be made in a polypeptide can be based on the analysis of amino acid change frequencies between homologous proteins of different species, as shown, for example, in Table 12, Schulz et al., G.E. Principles of Protein Structure, Springer-Verlag, New York, NY, 1798, and in Figure 3-9, Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman and Co., San Francisco, CA 1983. According to such an analysis, conservative substitutions are defined herein as exchanges in one of the following five groups:

Table IB

1. Small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr (Pro, Gly);

2. Poorly negatively charged residues and their amides: Asp, Asn, Glu, Gln;

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

4. Large aliphatic non-polar residues: Met, Ile, Leu, Val, (Cys); and

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

The three amino acid residues shown in parentheses in the table have a special role in the protein structure. Gly is the only residue without any side chain and thus causes chain flexibility. However, it also tends to form a secondary structure other than α-helix. Pro, due to its unusual geometry, significantly reduces the chain shape and usually supports the formation of β-thread-like structures, although in »· φ φ

• In some cases, Cys may be able to participate in the formation of disulfide bonds that are important for protein folding. Note that Schulz et al., Supra, merges groups 1 and 2. Also note that Tyr, due to its hydrogen bonding potential, has a significant association with Ser and Thr, etc.

The conservative amino acid substitutions of the present invention, as outlined above, are known in the art and are believed to preserve the biological and structural properties of the polypeptides upon amino acid substitution. Most of the deletions and substitutions of the present invention are those that do not cause radical changes in the characteristics of the protein or polypeptide molecule. Characteristics indicate both changes in secondary structure, such as α-helix or β-sheet, as well as changes in biological activity, such as binding to RIP and / or mediating the effects of RIP on cell death.

Examples of preparing amino acid substitutions in proteins that can be used to obtain analogs of RAP-2 proteins for use in the present invention include any known methods as disclosed in U.S. Pat. RE 33653, 4959314, 4588585 and 4737462 (Mark et al.); 5,116,943 (Koths et al.); 4,965,195 (Namen et al.); 4,879,111 (Chong et al.); and 5017691 (Lee et al); and the lysine substituted proteins disclosed in U.S. Pat. No. 4,945,484 to Shaw et al.

In addition to the conservative substitutions listed above, which do not significantly alter the activity of the RAP-2 protein, the scope of the present invention includes conservative substitutions or less conservative and more random changes that result in increased biological activity of RAP-2 proteins.

· 9 9 • 99

9999 ·

9999 8 9

9

99

9 9 9

9

9 9 9

99

If it is desired to determine the exact effect of substitutions or deletions, one skilled in the art can evaluate the effect of such substitutions and deletions, etc. by routine binding and cell survival assays. Investigations using such standard tests do not require unnecessary experimentation.

Acceptable RAP-2 analogs are those which retain at least the ability to bind to RIP and therefore, as noted above, retain the ability to mediate RIP activity on the intracellular signal transduction pathways as described above. In this way, analogs having a so-called dominant-negative effect can be prepared, in particular analogs which are defective either in binding to RIP or in subsequent signal transmission or in other activities following such binding. Such analogs can be used, for example, to inhibit the effect of RIP, or to inhibit the inducing effect of RIP (direct or indirect) on NF-κΒ, depending on which of these activities are the major activities affected by the RIP-RAP-2 interaction (see above) thus, such analogs can compete with natural RAP-2 for binding to RIP.

At the genetic level, these analogs are usually prepared by site-directed mutagenesis of nucleotides in the DNA encoding the RAP-2 protein, thereby producing the DNA encoding the analog, and then synthesizing the DNA and expressing the polypeptides in recombinant cell culture. Analogs typically exhibit the same or increased qualitative biological activities as the native protein. (Ausubel et al., Current Protocols in Molecular Biology,

Greene Publications and Wiley Interscience, New York, NY, 1987-1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

4 9 4 4 44 44

The preparation of the RAP-2 protein of the present invention, or an alternative nucleotide sequence encoding the same polypeptide but differing from the native sequence due to changes due to known degeneracy of the genetic code, can be accomplished by site-specific mutagenesis of DNA encoding an previously prepared analog or native version. RAP-2 protein. Site-directed mutagenesis allows for the production of analogs using specific oligonucleotide sequences that encode a DNA sequence with the desired mutations, as well as a sufficient number of contiguous nucleotides, to obtain an average sequence of sufficient size and complexity to form a stable duplex on both sides of the deletion to be bridged. Typically, a primer of about 20 to 25 nucleotides in length, with about 5 to 10 complementary nucleotides on each side of the sequence being altered is used. Generally, the site-specific mutagenesis technique is well known in the art, as disclosed, for example, in Adelman et al., DNA 2: 183 (1983), the disclosures of which are incorporated herein by reference.

As is known, site-specific mutagenesis techniques typically utilize phage vectors that exist in both single-stranded and double-stranded forms. Typical vectors useful in site-directed mutagenesis are vectors such as M13 phage as described in Messing et al., Third Cleveland Symposium on

Macromolecules and Recombinant DNA, ed. A. Walton, Elsevier, Amsterdam (1981), the disclosure of which is incorporated herein by reference. These phages are readily available commercially and their use is known to those skilled in the art. Alternatively, plasmid vectors that contain a single-stranded phage sequence recognizing the origin of replication (Veira et al., Meth. Enzymol. 153: 3, 1987) can be used to obtain single-stranded DNA.

In general, site-directed mutagenesis according to the present tt

4444 4 994 444 94 44 of the invention is first performed by preparing a single-stranded vector comprising in its sequence a DNA sequence that encodes the respective polypeptide. The oligonucleotide primer carrying the desired mutated sequences is prepared by synthetically automated DNA / oligonucleotide synthesis. This primer is then heat-treated with a single-stranded vector containing the protein sequence and then treated with DNA-polymerizing enzymes, such as the E. coli Klenow fragment of polymerase I, which completes synthesis of the mutation-containing chain. Thus, the mutated sequence and the second strand carry the desired mutation.

This heteroduplex vector is then used to transform suitable cells, such as E. coli JM101, and clones containing recombinant vectors carrying the mutated sequences are selected.

Upon selection of such a clone, the mutated RAP-2 protein sequence can be removed and placed in a suitable vector, generally a transfer or expression vector of the type that can be used to transfect a suitable host.

Also, a gene or nucleic acid encoding a RAP-2 protein may be detected, obtained and / or modified - in vitro, in situ and / or in vivo, using known DNA or RNA amplification techniques, such as PCR or chemical oligonucleotide synthesis. PCR allows the amplification (increase in number) of specific DNA sequences by repeated reactions with DNA polymerase. This reaction can be used as a replacement for cloning; all that is needed is knowledge of the nucleic acid sequence. For PCR, primers that are complementary to the selected sequence are designed. 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 they can be tolerated.

9 9 9

· Errors in pairing of complementary bases. Amplification of these error regions can lead to the synthesis of mutated products that lead to the generation of peptides by novel properties (i.e., site-directed mutagenesis). See also Ausubel, supra, chap.

16. Also, when combining complementary DNA synthesis (cDNA) using reverse transcriptase, with PCR, RNA can be used as a starting material for synthesis of the extracellular domain of the prolactin receptor without cloning.

Further, PCR primers can be designed to insert new restriction sites or other properties, such as termination codons, at the end of the gene segment being amplified.

This positioning of the restriction sites at the 5 'and 3' ends of the amplified gene sequence allows the gene segments encoding the RAP-2 protein or fragment thereof to be adapted to be ligation to other sequences and / or cloning sites in the vector.

PCR and other methods of amplifying RNA and / or DNA are well known in the art and can be used in the present invention without undue experimentation, according to the description and guidance provided herein. Known DNA and RNA amplification techniques include, but are not limited to, polymerase chain reaction (PCR) and similar amplification processes (see, eg, US Patent Nos. 4683195, 4683202, 4800159, 1965188 (Mullis et al .; 4795699 and 4921794, Tabor et al.) 5142033 (Innis); 5122464 (Wilson et al.); 5091310 (Innis); 5066584 (Gyllensten et al.); 4889818 (Gelfand et al.); 4994370 (Silver et al.); 4766067 (Biswas); 4656134 (Ringold); and Innis et al., Eds., PCR Protocols: A Guide to Method and Applications) and RNA-mediated amplification that uses antisense RNA to target a template as a double-stranded DNA synthesis template (US Patent No. 5,130,238 to Malek et al. al.), with the trade name tf tf tf tf tf tf tf tf tff tff tff tff tff tff tff tff tff

NASBA); and immuno-PCR that combines the use of DNA amplification with antibody labeling (Ruzicka et al., Science 260: 487 (1993); Sano et al., Scince 258: 120 (1992); Sano et al., Biotechniques 9: 1378 ( 1991)), wherein the entire contents of these patents and publications are incorporated herein by reference.

Biologically active fragments of RAP-2 proteins (e.g., any RAP-2 or isoform thereof) can be prepared in an analogous manner. Suitable fragments of RAP-2 proteins are those fragments that retain the properties of RAP-2 and which can directly or indirectly affect or mediate the biological activity of RIP or other RIP-associated proteins. Accordingly, fragments of the RAP-2 protein can be prepared having a dominant-negative or dominant-positive effect as defined above for the analogs. It will be appreciated that these fragments represent a special class of analogs of the present invention, in particular, are defined portions of RAP-2 proteins derived from the complete sequence of the RAP-2 protein (e.g., any RAP-2 or isoforms thereof). the fragment has any of the above desirable activities. Such a fragment may be, for example, a peptide.

Similarly, derivatives may be prepared by standard modification of the side groups of one or more amino acid residues of the RAP-2 protein, analogs and fragments thereof, or by conjugating the RAP-2 protein, analogs or fragments thereof to another molecule, for example antibodies, enzyme, receptor, etc. as is well known in the art. The term derivatives as used herein includes derivatives that can be prepared from functional groups present as side chain residues or N- or C-terminal groups, using techniques known in the art, and such derivatives are within the scope of

9 *

• · • ·

9 9

989 99 9

9 9 9

99999 · 9 9

9999 9 999 9 of the present invention. Derivatives may contain chemical groups such as carbohydrate or phosphate residues, provided that such a fraction has the same or higher biological activity as the RAP-2 proteins.

For example, derivatives may include aliphatic carboxylic acid esters, carboxylic amides formed by reaction with ammonia or primary or secondary amines, N-acyl derivatives, or free amino groups of amino acid residues formed with acyl groups (e.g., an alkanoyl or carbocyclic aroyl group) or O- acyl derivatives of free hydroxyl groups (e.g., a seryl or threonyl radical) formed with acyl groups.

The term derivative includes only those derivatives that do not change one amino acid for another of the twelve common natural amino acids.

RAP-2 is a protein or polypeptide, i.e., amino acid residue sequences. A polypeptide consisting of a longer sequence that comprises the entire sequence of the RAP-2 protein, in accordance with the definitions herein, is within the scope of the present invention as long as the additions do not affect the basic and novel characteristics of the invention, i.e. can be cleaved to form a protein or polypeptide having the biological activities of the RAP-2 protein. Thus, the present invention includes, for example, fusion proteins consisting of the RAP-2 protein and other amino acids or peptides.

The new RAP-2 protein, its analogs, fragments and derivatives, have many different uses, such as:

· 0 · 0 0 0 0 0 0 0 0 0 0

(I) The RAP-2 protein, analogs, fragments and derivatives thereof can be used to affect RIP function in the signal transduction pathways leading to inflammation, cell death, or cell survival as described above. For example, if RAP-2 can affect the effect of RIP on NF-κB, JNK (Jun kinase) or p38 kinase activation, both such RAP-2 effects leading to potentiation of RAP-2-RIP effects can be used for anti-tumor, anti- and pro-inflammatory, anti-HIV applications and the like. In this case, the RAP-2 protein, analogs, fragments or derivatives thereof that affect inflammation, enhance cytotoxic effects, or block cell survival effects can be introduced into cells using standard procedures. For example, if the RAP-2 protein is completely intracellular (as expected) and is to be introduced only into cells in which the RIP-mediated effect of FAS-R ligands or TNF or other cytotoxic protein is desired, a system for specific protein insertion is required. into cells. One way to make this insertion is. generating a recombinant animal virus, such as a vaccinia-derived virus, into which DNA has been inserted for the following two genes: a gene encoding a ligand that binds to cell surface proteins specifically expressed by cells, such as an HIV gp120 protein that specifically binds to some cells (CD4 lymphocytes and related leukemia cells) or any other ligand that binds specifically to cells carrying FAS-R or p55-R, so that the recombinant viral vector is capable of binding to such cells carrying FAS-R or p55-R; and a gene encoding the RAP-2 protein. Thus, the expression of a surface binding protein on the surface of a virus specifically binds the virus to tumor cells or other cells carrying FAS-R or p55-R, and then the sequence encoding the RAP-2 protein is inserted into the cells via the virus and leads to effect FAS-R ligands or TNF mediated by RIP or RIP independent.

* · · · · · · · · · · · · · · · · · · · ·

198:

• · · · ···· · · ···· ·

The construction of such recombinant animal viruses is a standard procedure (see, for example, Sambrook et al.,

Another possibility is to insert RAP-2 protein sequences (e.g., any RAP-2 or isoforms thereof) in the form of oligonucleotides that can be absorbed by the cells and expressed in the cells.

(ii) They can be used to inhibit the effects of FAS-R ligand or TNF or related proteins that are mediated by RIP or RIP independent, for example in cases such as tissue damage in septic shock, graft versus host disease or acute hepatitis in which blocking of FAS-R ligand or TNF-induced FAS-R or p55R triggered intracellular signal transduction or RIP independent effects, or other protein-mediated signal transduction, while desirable enhancement of cell survival signaling pathways is desirable. In this situation, for example, it is possible to insert into cells - using standard techniques, oligonucleotides having an antisense coding sequence for the RAP-2 protein that effectively block the translation of mRNA encoding the RAP-2 protein and thereby block its expression and lead to FAS-R ligand , TNF, RIP or other proteins. Such oligonucleotides may be introduced into cells using the above recombinant virus approach, wherein the second sequence in the virus is an oligonucleotide sequence.

Similarly, as noted above, depending on the nature of the RAP-2-RIP interaction, methods (i) and (ii) above may enhance or inhibit signaling pathways leading to cell inflammation or survival.

Another possibility is to use antibodies specific for the RAP-2 protein to inhibit its intracellular signaling.

• · · · · · · · · · · · · · · · · · ·

9

9999 8 9

Yet another method for inhibiting RIP-mediated effects or RIP independent effects is the newly developed ribozyme technique. Ribozymes are catalytic RNA molecules that specifically cleave RNA. Ribozymes can be engineered to cleave a selected target RNA, for example, an mRNA encoding the RAP2 protein of the present invention. Such ribozymes will have mRNA-specific sequences for the RAP-2 protein and will be able to interact with these sequences (complementary binding), followed by mRNA cleavage that will result in a decrease (or complete loss) of RAP-2 protein expression, where it is dependent on the level of ribozyme expression in the target cells. Any suitable vector, for example a plasmid, animal viral (retroviral) vectors, which are typically used for this purpose (see (i) above), can be used to introduce ribozymes into selected cells (e.g., cells carrying FAS-R or p55-R). the virus contains, as a second sequence, a cDNA encoding the selected ribozyme sequence). (For a review of methods related to ribozymes, see Chen et al., 1992; Zhao and Piek, 1993; Shore et al., 1993; Joseph and Burke, 1993; Shimayama et al., 1993; Cantor et al., 1993; Baringa, 1993; Crisell et al., 1993 and Koizumi et al., 1993). This approach is useful when the RAP-2-RIP interaction increases cellular cytotoxicity in situations where it is desirable to block this cytotoxicity, or when the RAP-2-RIP interaction inhibits NF-κΒ activation in the same situation when it is desirable to block this inhibition to increase such NF-κΒ activation, ie in both cases it is desirable to increase cell survival, as in (ii), above.

(iii) The RAP-2 protein, analogs, fragments and derivatives thereof can also be used to isolate, identify and clone other proteins of the same class, ie, proteins binding to RIP or functionally related receptors and proteins involved in intracellular signal transduction processes. . In this application, the aforementioned yeast two-hybrid system may be utilized, or a newly developed system using Southern hybridization under low stringency conditions may be used, followed by PCR cloning (Wilks et al., 1989). In Wilks et al. discloses the identification and cloning of two putative proteintyrosine kinases by Southern hybridization under low stringency conditions, followed by PCR cloning based on the knowledge of the kinase motif sequence, part of the kinase sequence. This approach can be used, in accordance with the present invention, using the sequence of the RAP-2 protein, to identify and clone related RIP-binding proteins.

(iv) Yet another way of utilizing the RAP-2 protein, analogs, fragments and derivatives thereof of the present invention is their use in affinity chromatography methods to isolate and identify other proteins or factors that are capable of binding to other proteins or factors involved in the processes intracellular signal transduction. In this application, the RAP-2 protein, analogs, fragments and derivatives thereof can be individually bound to affinity chromatography matrices and then contacted with cell extracts or isolated proteins or factors suspected to be involved in intracellular signal transduction processes. After affinity chromatography, other proteins or factors that bind to the RAP-2 protein, or analogs, fragments or derivatives thereof of the present invention, can be eluted, isolated, and characterized.

(v) As mentioned above, the RAP-2 protein, analogs, fragments, and derivatives thereof of the present invention can also be used as immunogens (antigens) for production. · Specific antibodies directed against these antigens. · · Specific antibodies directed against these antigens. These antibodies can also be used to purify the RAP-2 protein (e.g., RAP-2 or any isoform thereof) either from cell extracts or from transformed cell lines producing the RAP-2 protein or analogs or fragments thereof.

Further, these antibodies can be used for diagnostic purposes to identify diseases related to the normal function of a FAS-R ligand or TNF system mediated by RIP or independent of RIP activity, for example, excessive or decreased effects induced by FAS-R ligand or TNF mediated RIP or RIP effects. per cell. In such diseases associated with a malfunction of an intracellular signal transduction system involving a RIP protein, or other, the above-mentioned RIP-binding proteins, or the RAP-2 protein itself, such antibodies may serve as a significant diagnostic tool.

It should also be noted that the isolation, identification and characterization of the RAP-2 protein of the present invention can be performed using well known screening techniques. For example, one of these techniques, the yeast two-hybrid system as described below, was used to identify the RIP protein (see Stanger et al., 1995) and subsequently the various RAP-2 proteins of the present invention (among other novel proteins mentioned above and below). in related patent applications). Similarly, other procedures such as affinity chromatography, DNA hybridization, etc., as well as other well-known methods in the art by which the isolation, identification and characterization of the RAP-2 protein of the present invention can be carried out, may be used or isolating, identifying, and characterizing other proteins, factors, receptors, etc. that are capable of binding to the RAP-2 proteins of the present invention.

As mentioned above, the RAP-2 protein can be used to generate antibodies specific for RAP-2 proteins, for example, for RAP-2 and its isoforms. The antibodies or fragments thereof may be used as detailed below, wherein in these applications the antibodies or fragments thereof are specific for RAP-2 proteins.

Based on the finding that RAP-2 binds specifically to RIP and is therefore a mediator / modulator of RIP, it can influence / mediate RIP activity in signal transduction pathways leading to inflammation, cell death or cell survival in which RIP-2 participates independently or together with other proteins (for example, FAS-R, p55-R, MORT-1, MACH, Mch4, G1, and TRADD in cell death pathways, or with TRAF2 in cell survival pathways), and therefore it is important to develop drugs that can potentiate or inhibit the RAP-2-RIP interaction, as appropriate, and which of these signal transduction pathways are amplified / inhibited by the RAP-2-RIP interaction. There are many diseases in which such drugs can be of great benefit. Such diseases include, for example, acute hepatitis in which acute liver injury reflects FAS-R ligand-mediated hepatic cell death; autoimmune-induced cell death, such as pancreatic pancreatic β Langerhans cell death, which is the cause of diabetes; cell death in transplant rejection (e.g., kidney, heart or liver); brain death of oligodendrocytes in multiple sclerosis; and HIV-inhibited T-cell death that causes HIV virus proliferation and therefore AIDS.

It is possible that RAP-2 or one or more of its possible isoforms may serve as a natural inhibitor of RIP in one •

It may also be used as a specific inhibitor of RIP. Similarly, other substances, such as peptides, organic compounds, antibodies, etc., can also be screened to obtain specific drugs that can inhibit RAP-2-RIP interaction.

An example of how peptide inhibitors of the RAP-2-RIP interaction can be designed and investigated is based on previous studies of peptide inhibitors of ICE or ICE-like proteases, ICE substrate specificity, and strategies for epitope analysis by peptide synthesis. The minimum requirement for efficient cleavage of the ICE peptide has been found to be four amino acids to the left of the cleavage site with a strong preference for aspartic acid at the P1 position and with methylamine to the right of the P1 position (Sleath et al., 1990; Howard et al., 1991;

Thornberry et al., 1992). Furthermore, it was found that the fluorogenic substrate peptide (tetrapeptide) acetyl-Asp-Glu-Val-Asp-α (4-methyl-cumaryl-7-amide) (Ac-DEVD-AMC) corresponding to the sequence in the (ADP-ribose) polymerase ( PARP) is cleaved in cells shortly after FAS-R stimulation, as well as in other apoptotic events (Kaufmann, 1989; kaufmann et al., 1993; Lazebnik et al., 1994) and that it is effectively cleaved by CPP32 (member of CEEED3 / ICE family) proteas) and MACH proteases (and probably also Gl-proteases - see, for example, related IL 120367).

Since Asp appears to be significant at the P1 position of the substrate, tetrapeptides containing Asp as the fourth amino acid residue and various combinations of amino acids in the first three positions can be rapidly examined for binding to the active site of the protease using, for example, the method described by Geysen (Geysen, 1985) (Geysen et al., 1987), in which a large number of peptides on solid supports are screened for specific interactions with antibodies. The binding of MACH proteases to specific peptides can then be detected using a variety of well known detection methods, such as radiolabeling G1 proteases and the like. This Geysen method has been shown to allow testing of at least 4000 peptides every working day.

Similarly, the exact binding region or homology region that determines the interaction between RAP-2 and RIP can be analyzed, and peptides that block this interaction can be screened, for example, peptides synthesized to have a similar sequence to that of the binding region or complementary sequence. to a binding region that competes with natural RAP-2 for binding to RIP.

Drugs or peptide inhibitors that are capable of inhibiting the inflammatory or lethal activity of RAP-2 by inhibiting the RAP-2-RIP interaction may also be conjugated to, or complexed with, molecules that facilitate cell entry.

U.S. Pat. U.S. Pat. No. 5,149,782 discloses the conjugation of molecules to be transported across a cell membrane with membrane-confluent agents such as fusogenic polypeptides, ion channel forming polypeptides, other membrane polypeptides, and long-chain fatty acids such as myristic acid or palmitic acid. These membrane blending agents insert the molecular conjugate into the lipid bilayer of cell membranes and facilitate their entry into the cytoplasm.

Low et al. No. 5108921, reviews available methods for transmembrane transfer of molecules such as proteins and nucleic acids through the mechanism of receptor-mediated endocytotic activity. These receptor systems include systems 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. disclose that receptors for nutritional compounds such as biotin and folate receptors can be advantageously used to enhance cell membrane transport due to the localization and abundance of biotin and folate receptors on most cell surface membranes and associated receptor-mediated transmembrane transfer mechanisms. Thus, the complex formed between the compound to be delivered to the cytoplasm and a ligand such as biotin or folate is contacted with a cell membrane bearing biotin or folate receptors to initiate receptor-mediated transmembrane transfer mechanisms that allow the selected compound to enter the cell.

Further, it is known in the art that fusing a selected peptide sequence to a leader / signal peptide sequence creates a chimeric peptide that can be transported across the cell membrane into the cytoplasm.

As will be appreciated by those skilled in the art of peptide, the peptide inhibitors of the RAP-2-RIP interaction of the present invention include peptidomimetic drugs or inhibitors that can also be rapidly screened for binding to the RAP-2 / RIP protease to produce even more stable inhibitors.

It will also be appreciated that the same means as mentioned above for facilitating or enhancing the transport of peptide inhibitors across cell membranes are also applicable to RAP-2 or its isoforms themselves, as well as *.

9 9 9 9 · 9 · • 9 9 9 9 for other peptides and proteins that exert their effects intracellularly.

The term antibody as used above refers to polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic antibodies (anti-Id) to antibodies that can be labeled in soluble or bound form, as well as fragments thereof obtained by any known method. by a technique such as, for example, enzymatic cleavage, peptide synthesis, or a recombinant technique.

Polyclonal antibodies are a heterogeneous population of antibodies obtained from the sera of animals immunized with an antigen. The monoclonal antibody comprises a substantially homogeneous population of antigen-specific antibodies, wherein the population comprises significantly similar epitope binding sites. Mabs can be obtained by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature, 256: 495-497 (1975); U.S. Pat. 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 are incorporated herein by reference.

Such antibodies may be immunoglobulins of any class, including IgG, IgM, IgE, IgA, GILD, and any subclasses thereof. The hybridoma producing the mAbs of the present invention can be cultured in vitro, in situ or in vivo. Production of high titers of mAbs in vivo or in situ allows this presently preferred method of production.

Chimeric antibodies are molecules in which different parts originate from different animal species, such as molecules having a variable region from a mouse mAb and a human. Immunoglobulin constant region. 9 4 4 Immunoglobulin constant region. Chimeric antibodies are primarily used to reduce immunogenicity upon administration and to increase yield in preparation, for example, when the murine mAb has a higher hybridoma yield but a higher immunogenicity in humans, the human / mouse chimeric mAb is used. Chimeric antibodies and methods for their production are known in the art (Cabily 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 Nov. 14, 1984); Neuebrger et al., Nature 314: 268-270 (1985); Taniguchi et al., European Patent Application 171496 (published Feb. 19, 1985); Morrison et al., European Patent Application 173494 (published Mar 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. WO 8702671 (published May 7, 1987); Liu et al., Proc. Nati. Acad. Sci. USA 84: 34393443 (1987); Sun et al., Proc. Nati. Acad. Sci. USA 84: 214218 (1987); Better et al., Science 240: 1041-1043 (1988); and

Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, supra. These references are incorporated herein by reference in their entirety.

An anti-idiotypic (anti-Id) antibody is an antibody that recognizes unique determinants commonly associated with an antibody binding site for an antigen. An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., a mouse strain) as the source of the antibodies to which the anti-Id is to be prepared. The immunized animal will recognize and respond to idiotypic determinants of the immunizing antibody and will produce antibodies to these idiotypic determinants (anti-Id antibodies). See, for example, U.S. Pat.

No. 444,480, the disclosure of which is incorporated herein by reference.

The anti-Id antibody can also be used as an immunogen to induce an immune response in yet another animal that then produces so-called anti-anti-Id antibodies. Anti-anti-Id antibodies can be epitope-identical to the original mAbs that induced anti-Id. Thus, other clones expressing antibodies of identical specificity can be identified by using antibodies to idiotypic determinants of the mAb.

Accordingly, mAbs against the RAP-2 proteins, analogs, fragments and derivatives thereof of the present invention can be used to induce anti-Id antibodies in suitable animals such as BALB / c mice. Spleen cells from such immunized mice can be used to prepare anti-Id hybridomas secreting anti-Id mAbs. In addition, anti-Id mAbs can be coupled to a carrier such as sting hemocyanine (KLH) and can be used to immunize other BALB / c mice. Serum from these mice will then contain anti-anti-Id antibodies having the binding properties of the original mAbs specific for the epitope of the above RAP-2 protein or analogs, fragments and derivatives thereof.

Anti-Id mAbs therefore have their own idiotypic epitopes, or idiotopes structurally similar to evaluated epitopes, such as GRB protein-α.

The term antibody also includes both intact molecules and fragments thereof, such as, for example, Fab and F (ab ') 2, which can bind to an antigen. Fab and F (ab ') 2 fragments do not contain an Fc fragment of intact antibody, are removed more rapidly from circulation and may have less specific tissue binding than intact antibody (Wahl et al., J. Nucl. Med. 24: 316-325). 9 9 9 (1983).

It will be appreciated that Fab and F (ab ') 2 and other antibody fragments useful in the present invention can be used to detect and quantify the RAP-2 protein using the methods described for intact antibodies. Such fragments are usually prepared by proteolytic cleavage, using enzymes such as papain (to prepare Fab fragments) or pepsin (to prepare F (ab ') 2 fragments.

An antibody is capable of binding to a molecule if it can specifically react with the molecule and thus bind the molecule to the antibody. The term epitope refers to that portion of any molecule capable of binding to an antibody that can also be recognized by the antibody. Epitopes or antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and have specific three-dimensional structural characteristics as well as a specific charge.

An antigen is a molecule or portion of a molecule that can bind to an antibody and which can further induce the production of antibodies capable of binding to an epitope of an antigen in an animal. An antigen may have one or more than one epitope. The specific reaction described above means that the antigen reacts, in a highly selective manner, with the respective antibody and not with other antibodies that may be elicited by other antigens.

The antibodies, including antibody fragments, useful in the present invention can be used to quantitatively or qualitatively detect the RAP-2 protein in a sample or to detect cells that express the RAP-2 protein of the present invention. This can be done using • * • · · (Λ

Immunofluorescence techniques using fluorescently labeled antibodies (see below) along with light microscopy, flow cytometry, or fluorometric detection.

The antibodies (or fragments thereof) useful in the present invention can be used histologically, for example, in immunofluorescence or immunoelectron microscopy, to detect the RAP-2 protein of the present invention in situ. In situ detection may be performed by collecting a histological sample from a patient and applying the labeled antibody of the present invention to such a sample. Preferably, the antibody (or fragment thereof) is applied or coated as a labeled antibody (or fragment thereof) to a biological sample. With such a procedure, it is possible to determine not only the presence of RAP-2 protein, but also its distribution in the tissue examined. It will be understood by those skilled in the art that, using the present invention, various histological methods (such as histological staining) may be modified to perform such in situ detection.

Such RAP-2 protein assays of the present invention typically comprise incubating a biological sample such as biological fluid, tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells that have been incubated in tissue culture in the presence of a detectably labeled antibody capable of identifying the RAP-2 protein, and detecting such an antibody by any technique known in the art.

The biological sample may be treated with a solid phase carrier or a carrier such as nitrocellulose, or with another carrier that allows the immobilization of cells, cell particles, or soluble proteins. The carrier may then be rinsed with a suitable buffer followed by administration of the detectably labeled antibody of the present invention as described above. The solid support may then be washed a second time with buffer to remove unbound antibody. The amount of label on said solid support can then be detected by conventional means.

The terms solid carrier, solid phase carrier, carrier include any carrier capable of binding antigen or antibodies. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural or modified celluloses, polyacrylamides, gabbro and magnetite. For purposes of the present invention, the carrier may be partially soluble or insoluble. The carrier material may have substantially any possible structural configuration as long as the bound molecule is capable of binding antigen or antibody. Thus, the carrier configuration may be spherical, as is the case with beads, cylindrical, as is the case with the inner surface of the test tube or the outer surface of the rod.

Alternatively, the surface may be flat, as is the case with sheets, test strips, etc. Preferred carriers are polystyrene strips. Those skilled in the art will be aware of, or will be able to identify such carriers without undue experimentation, other suitable carriers for binding the antibody or antigen.

The binding activity of a given batch of antibodies of the present invention as described above can be determined using well known methods. Those skilled in the art are able to select functional and optimal test conditions for each assay using routine experiments.

The test may also include other steps such as washing, mixing, shaking, filtering and the like, if any, or if desired; ί necessary in a certain situation.

One way in which an antibody of the present invention can be detectably labeled is by binding the antibody to an enzyme and using the enzyme immunoassay (EIA). This enzyme, when exposed to a suitable substrate, reacts with the substrate in such a way that a chemical moiety is formed which can be detected, for example, spectrophotometrically, fluorometrically or visually. Enzymes that can be used for detectable labeling of an antibody include, for example, malat dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate dehydrogenase, triosaphosphate isomerase, horseradish peroxidase, alkaline phosphatase , asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine esterase. Detection can be accomplished by colorimetric methods that utilize a chromogenic substrate for the enzyme. Detection can also be performed by visual comparison of the extent of the enzymatic reaction of the substrate with similarly prepared standards.

Detection can be performed using many other immunoassays. For example, RPTPases can be detected by radiolabeling antibodies or antibody fragments by radioimmunoassay (RIA). A good description of RIA is given in Laboratory Techniques and Biochemistry in Molecular Biology, Work, T.S. et al., North Holland Publishing Company, NY (1978), in particular in the chapter entitled An Introduction to Radioimmune Assay and Related Technique, Chard, T., which is incorporated herein by reference. The radioactive isotope can be detected by a gamma camera or scintillation camera or by autoradiography.

• »

9 9 9 · · 9 9 9 9 9 * 9 ·

99 «»

9,999 9 ·

9 · «·· · 9 99

It is also possible to label the antibodies of the present invention with fluorescent compounds. When the fluorescently labeled antibody is exposed to light of the correct wavelength, it can be detected by fluorescence. The most commonly used fluorescently labeled compounds include fluorescein isothiocyanate, rhodamine, phycoerythrine, pycoocyanine, allophycocyanine, o-phthaldehyde and fluorescamine.

The antibody may also be detectably labeled using fluorescent emitting metals such as 152E or other lanthanide series metals. These metals can be bound to the antibody by metal chelating groups such as diethylenetriaminepentaacetic acid (ETPA).

The antibody may also be detectably labeled by binding to a chemiluminescent compound. The presence of the antibody bound to the chemiluminescent compound is then determined by detecting the presence of luminescence that occurs during the chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

Similarly, 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 the catalytic system increases the efficiency of the chemiluminescent reaction. The presence of the bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for labeling purposes are luciferin, luciferase and aequorin.

• »

4 4

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4 4

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4 9.

4 4 * *

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

Typical and preferred immunotherapeutic assays include sequential assays in which the antibody bound to a solid support is first contacted with a test sample for extracting the antigen from the sample by forming a binary complex formed between the solid phase antibody and the antigen. For a suitable incubation period, the solid support is washed to remove the remainder of the liquid that contains unreacted antigen and then contacted with a solution containing an unknown amount of labeled antibody (which acts as a reporter molecule). After a second incubation period allowing the complexing of the labeled antibody with the antigen bound to the solid support via the unlabeled antibody, the solid support is washed a second time to remove unbound labeled antibody.

In another type of sandwich test, which can also be used with the antigens of the present invention, a so-called simultaneous and reverse test is used. The simultaneous assay includes an Eden incubation step because the antibody bound to the solid support and the labeled antibody are both added to the test sample at the same time. After the incubation is complete, the solid support is washed to remove residual liquid sample and unbound labeled antibody. The presence of the labeled antibody associated with the solid support is then determined in the same manner as in the conventional sequential sandwich test.

In the reverse assay, the first solution containing the labeled antibody is first added to the test liquid sample, and then, after a suitable incubation period, the unlabeled antibody bound to the solid support is added. After the second incubation period, the solid phase is washed in a conventional manner to remove the free residual liquid sample and unbound labeled antibody. The presence of labeled antibody associated with the solid support is then determined in the same manner as in a conventional simultaneous and sequential sandwich assay.

The RAP-2 proteins of the present invention can be produced by any standard recombinant DNA technique (see, for example, Sambrook et al., 1989, and Ansabel et al., 19871995, supra) in which suitable prokaryotic or eukaryotic host cells well known in the art are transformed. suitable prokaryotic or eukaryotic vectors containing protein coding sequences. Accordingly, the present invention also relates to such expression vectors and transformed hosts for the production of proteins of the present invention. As mentioned above, these proteins also include biologically active analogs, fragments and derivatives thereof, and hence, such vectors include vectors encoding analogs and fragments of these proteins, and transformed hosts include hosts producing such analogs and fragments. Derivatives of these proteins produced by transformed hosts are derivatives produced by standard modification of proteins or analogs or fragments thereof.

The present invention also relates to pharmaceutical compositions comprising recombinant animal viral vectors encoding

44 44

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445 49 t * «* *» 9 t

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RAP-2 proteins, wherein these vectors also encode a viral surface protein capable of binding to surface proteins of specific target cells (e.g., tumor cells), to allow insertion of sequences for RAP-2 proteins into these cells. Other pharmaceutical compositions of the present invention comprise as an active ingredient (a) an oligonucleotide sequence encoding an antisense sequence to the RAP-2 protein sequence, or (b) drugs that block the RAP-2-RIP interaction.

The pharmaceutical compositions of the present invention contain sufficient active ingredient to achieve the desired effect. Further, the pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers, including excipients and auxiliary agents, which facilitate processing of the active ingredient into a composition which can be used pharmaceutically and including excipients which can stabilize such compositions for administration to an individual in need of treatment as selected. expert in the field.

It is believed that the RAP-2 protein and its isoforms or isotypes are expressed in different tissues in varying amounts and also with a different isotype profile, as is the case with various other proteins involved in intracellular signal transduction pathways as described in the aforementioned related patent applications. These differences may contribute to the tissue-specific nature of the response to Fas1A1I-ligands and TNF. As with other CED3 / ICE homologs (Wang et al., 1994; Alnemri et al., 1995), the inventors have previously demonstrated (in the above-mentioned patent applications) that MACH isoforms that contain incomplete CED3 / ICE regions (e.g. MACHα3) have an inhibitory effect on the activity of co-expressed MACHα1 or MACHα2 molecules; they have also been found to block cell death induction.

Fas / APO1 and p55-R. Expression of such inhibitory isoforms in cells may constitute a mechanism of cellular self-defense against cyt / toxicity mediated by Fas / APO1 and TNF. A similar inhibitory effect of at least some of the G1 isoforms is also contemplated (G1 is a newly isolated novel Mch4- and perhaps a MACH binding protein that contains MORT modules and a protease domain - see related IL 120367). The considerable heterogeneity of the MACH isoforms and the predicted analogous heterogeneity of the G1 isoforms, which significantly exceeds the heterogeneity observed with other CED3 / ICE family proteases, allows for a fine-tuning of the function of the active MACH3 isoforms and analogously of the active G1 isoforms. Therefore, as noted above, RAP-2 proteins or possible isoforms may have different effects on different tissues with respect to their interactions with RIP and affecting the balance between activation of cell death pathways or survival as described above.

It is also possible that some of the possible RAR-2 isoforms have other functions. For example, RAP-2 or some RAP-2 isoforms may act as binding sites for molecules that participate in other, non-cytotoxic effects of Fas / APO1 and TNF receptors through interaction with or independently of RIP.

Due to the unique ability of Fas / APO1 and TNF receptors to cause inflammation, cell death, as well as the ability of TNF receptors to trigger other tissue damaging activities, malfunctions of these receptors may be particularly harmful to the organism. In addition, both excessive and decreased function of these receptors have been shown to contribute to the pathological manifestations of various diseases (Vasalli, 1992; Nagata and Golstein, 1995). Identify molecules involved in signal transduction from receptors and discover ways to influence

The activity of these molecules can lead to new therapeutic approaches. Other aspects of the present invention will be described in the following examples.

The invention will now be described in more detail in the following examples and the accompanying drawings.

It should be noted that the procedures:

(i) a two-hybrid screening and two-hybrid β-galactosidase expression assay; (ii) induced expression, metabolic labeling and immunoprecipitation of proteins; (iii) in vitro binding;

(iv) cytotoxicity evaluation; and (v) Northern blotting and sequence analysis (see also Boldin et al., 1995b) 2, 3 (see also Boldin et al., 1996) and 4, below, relating to MORT-1 and MORT-1 binding protein (e.g., MACH) ), as well as the newly isolated G1 protein (see IL 120367), are also fully usable (with some modifications) for the corresponding isolation, cloning and characterization of RAP-2 and its possible isoforms of the present invention. These procedures are therefore designed to fully describe the same procedures used for the isolation, cloning, and characterization of RAP-2 of the present invention as described in detail in related Israeli patent applications Nos. 114615, 114986, 115319,

116588,117932 and 120367, as in the corresponding PCT Application No. PCT / US96 / 10521. Furthermore, the NIK protein and its role in NF-κB activation and thus in cell survival and the role of TRAF2 in this cell survival, for example the interaction between TRAF2 and RIP and other proteins, have been described by the inventors in related IL 117800, IL 119133 and Malinin et al. , 1997.

9 9 9 9 9 9 9 9 9

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Cloning and isolation of RAP-2 protein that binds to RIP protein

Two-hybrid search, sequencing and preliminary analysis

Using a two hybrid search with RIP as capture reagent (see, for example, Fields and Song, 1989, WO / 96/18641) in a B-cell library, a clone of approximately 1.5 kb was isolated. This 1.5 kb clone (see arrow in Figs. 1 and 2) was used to screen the cDNA phage library, yielding an approximately 2.0 kb clone whose sequence is shown in Fig. 1.

EST pairing with a 1.5 kb clone sequence yielded an EST fragment that forms the 3 'end of the I.M.A.G.E consortium of clone # 41072 (Research Genetics Institute). From this clone, which originates from the fetal brain library, only two small fragments of the sequence and its 3 'and 5' ends are published. After obtaining the clone, this clone was sequenced and excluded if these published fragments contained errors, it was found that the sequenced clone (Fig. 2) was identical to the clone of Fig. 1 and its coding region, but showed differences in the 5'- non-coding region. Therefore, it is believed that both cDNAs are alternatively spliced forms of the same gene.

Sequence analysis has shown that, similar to RAP, the RAP-2 protein does not contain a lethal domain, does not contain a MORT module, does not contain a protease domain as proteins of the ICE family, does not contain a kinase domain, does not contain TRAF domains (for various domains present in intracellular signal transduction paths applications and various references, φ «in particular Malinin et al., 1997). Also, no significant motifs were present in the sequence, except for three leucine finger-like (LZ) blocks evenly distributed in the coding region of the protein. These blocks have been identified as similar because two of them contain Leu substitutions for Val, Met or Ile. Although such substitutions are generally considered to be conservative, it is unclear whether such substitutions in the leucine finger domain allow maintenance of the functional activity of the protein, i. links to other LZ. Binding studies have shown that RAP-2 binds to RIP and that RAP-2 is incapable of binding to TRADD, MORT-1, p55-R, p75-R, and MACH (in studies conducted so far). These results support the fact that RAP-2 does not contain deadly domains and MORT modules.

Therefore, RAP-2 appears to be a specific RIP-binding protein that interacts with / binds to RIP in a very specific manner. Thus, RAP-2 appears to be a specific modulator / mediator of the intracellular activity of RIP that plays a significant role in the RIP affecting / mediating signal transduction pathways leading to inflammation and cell death / survival.

Briefly, a RAP-2 clone was obtained by two-hybrid screening in the human B-cell cDNA library using the complete RIP protein as capture reagent. The RIP sequence was available from previous publications (e.g., Stanger et al., 1995) and is listed in the GenBank database under accession number U 25994 (human RIP sequence) (also the mouse RIP sequence - under accession number U 25995). According to this sequence, appropriate PCR-primers were designed using OLIGO4 ^(tra) software and the DNA fragment corresponding to the coding part of RIP was obtained by PCR using cDNA from the total human fibroblast RNA library as a template (using standard procedures). This coding portion of RIP was then cloned into the pGBT-9 vector (Clontech) and used as capture reagent as described above in a two-hybrid screening procedure. In this two-hybrid screen, a clone encoding a RIP binding protein that interacts with RIP was obtained.

This clone was used to screen the phage cDNA library and EST database. As seen in Figures 1 and 2, the coding sequences of the two clones are identical, while the 5'-non-coding regions are different. Thus, two alternatively spliced forms were probably obtained. The clones are about 2.0 kb in size with an ORF (open reading frame) of about 1.5 kb that determines the molecular weight of the protein itself of about 50 kd. The deduced amino acid sequence of RAP-2 is shown in FIG.

3.

Analyzes of the above RAP-2 clone sequences and sequences in the dbest database, the Human Genome Level 1 database, and the GenBank database showed that the RAP-2 sequence is a unique (new) sequence, since no known sequence shows any significant homology to this RAP-2. 2 sequences. Following the administration of IL 123758, which claims priority, Yamaoka S. et al. (1998) described the characterization of a mouse cDNA encoding a 48 kDa protein designated NEMO (according to the essential modulator NF-κΒ) (see prior art) .

Further database search (in silico) identified FIP2 - a protein of unknown function that was originally cloned by Li, Y. et al. (1998, see prior art).

As can be seen from the overall assembly of the RAP-2 and FIP-2 sequences (Figure 3), the degree of overall similarity is quite low (therefore it is not surprising that the sequence was not identified using global algorithm-based searches). Homology

99

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9 9 9

99

9

9 9 ·

9 9 ···· ♦ · • ·· · 9

Between RAP-2 and FIP-2 increases towards the C-terminus of the proteins and peaks at substantially identical C-terminal 30 amino acids. However, in addition to the last region, the putative LZ-motif FIP-2 (except for the Ile / Ala substitution) is greatly conserved in RAP-2.

Another shorter RAP-2 cDNA of approximately 0.5 kb (ID: 1469996) was also identified and was then designated as human shrt. The variant contains blocks of coding sequence derived from several distant regions of the 1.5 kb complete cDNA and is probably due to alternative splicing of the same gene.

Northern blot analysis was performed on a Multiple Tissue Northern blot (Clontech) with a 0.9 kb BglII-fragment of RAP-2 cDNA, which showed the complex character of RAP-2 mRNA. At least 5 different mRNAs were detected, ranging in size from <1 kb to> 7 kb, with a more or less marked prevalence of the 2.5 kb and 6 kb variants (Fig. 4A).

Example 2: Identification of murine RAP-2

A similar search of the mouse EST file in the TIGR revealed a partial cDNA of 1.6 kb (Mouse part. ID 761011, FIG.

3), which probably corresponds to murine RAP-2 because it is substantially identical (95%) to the human counterpart in the coding region (see FIG. 3).

However, the differences between human and mouse RAP-2 and NEMO exceed the differences that could be attributed to interspecific variations. Indeed, the missing block of 7 amino acids (position 249 at 20.4) from the mouse RAP-2 and NEMO sequence and insertion of 3 amino acids (KLE at position 111) in the NEMO open reading frame

99 99

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9

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9 compared to the complete human variant and partial mouse sequence are only the most visible examples (FIG. 3). However, these can lead to a functional effect on protein activity.

Indeed, the functions described for NEMO appear to be opposite to those described for human RAP-2, although fractionation analysis for NEMO confirmed that it is located in the signaloma.

Example 3: Binding of RIP to RAP-2 in mammalian cells

Further evidence of the biological significance of the RIP-RAP-2 interaction was obtained in transfected HEK-293T and HeLa cells. In addition, these proteins can be readily co-precipitated from cell lysates of HEK-293 (ATCC No. CRL 1573) cells transfected as follows in each lane in Fig. 4B and can be immunoprecipitated with an anti-FLAG mAb (Kodak). Immunocomplexes were then analyzed for the presence of HIS-RAP-2 by conventional western blotting with anti-His6 mAb (Sigma) (Fig. 4B and data not shown). However, the formation of such complexes does not result in RIP enzymatic activity: to the extent that we could verify the immunocomplex-kinase assay in vitro, it did not phosphorylate overexpressed RIP RAP-2 (not shown).

Binding assays were performed to determine whether RAP-2 binds to any other known intracellular signaling protein. In these assays, the TRADD, MORT-1, p55-R, p75-R, and MACH proteins were tested for binding to RAP-2. However, RAP-2 has been found to be incapable of binding to any of these proteins. RAP-2 also does not bind to any of the control proteins, such as lamin, cyclin D.

All of the above results indicate that the new RAP-2 protein is likely to interact with RIP in a very specific manner and as such represents a specific

• RIP modulator / mediator.

Example 4: RAP-2 interacts with NIK and affects NF-κB and c-Jun dependent transcription

Although no RAP-2-NIK interaction was detected in the two-hybrid yeast assay (see above), transfection experiments with HEK-293T mammalian cells indicated stable formation of this complex. The NIK-RAP-2 interaction was detected as in Example 3 except that the anti-FLAG antibodies were used for western blotting followed by immunoprecipitation with the anti-His6 antibody (Fig. 4C). This mismatch between yeast and mammalian cell binding was not surprising since complete NIK tends to lose its binding properties when expressed in yeast.

In view of the fact that both RIP and NIK appear to be indispensable mediators of TNF-induced NF-κB activation, we investigated whether over-expression of RAP-2 in cell culture is able to interfere with this particular signaling pathway. An initial set of experiments was performed on HEK-293T cells temporarily transfected with reporter plasmids containing the luciferase gene under the control of the HIV-LTR minimal promoter.

In a similar embodiment, RAP-2 was first found to reduce, almost to a baseline level, reporter gene activation caused by both overexpression of various known NFKB inducers involved in TNF-signaling (NIK, TRAF2, RIP, etc.) and external stimuli per cell (TNF and PMA, Fig. 5A). HEK-293T cells were transiently transfected with a reporter plasmid (HIVLTR-Luc or CMV-Luc for NF-κΒ (5A) and GAL4-Luc for c-Jun (5B) activation assays) and an expression vector for the respective inducer and either empty vehicle (pcDNA3) ) or a plasmid encoding complete RAP-2 (pcRAP-2). The fact that · · · · · · · · · · · · · · · ·

9 · · · · · · · · ···

RAP-2 may exert its effects as far in the signal transduction pathway as RelA suggests that part of this protein effect may be common to different and otherwise different signaling pathways (see below). At the same time, KB-independent (driven by the early CMV promoter) luciferase transcription was not disrupted (FIG.

5A) and therefore it is assumed that a possible generic disruption of the basic RAP-2 transcription / translation mechanisms can be avoided. These results were then fully confirmed in HeLa cells (not shown).

However, as titration tests have shown, the real phenomenon is much more complex. It was found that when TRAF2 was transiently expressed in HEK-293T cells together with different amounts of pcRAP-2 as shown in Fig. 6, RAP-2 significantly changed its behavior at low concentrations (about 20 ng / 10 ⁶ cells), when it enhanced TRAF2-induced NF-κΒ transcription (see Figure 6A). Further, by replacing the original insert with a reverse orientation insert, an efficient vehicle expressing the antisense sequence of RAP-2 was designed and TRAF2 was transiently expressed in HEK-293T cells along with various amounts of pcRAP2-a / s (antisense) constructs analyzed and the effect of progressive depletion of RAP- 2 and a concentration-dependency graph was obtained. The overall trend of the graph shows that the reactivity of the cells is roughly inversely proportional to the amount of transfected RAP-2 DNA, except for the 'zero point' characteristic region that belongs to the nominal, endogenous protein concentration (Fig. 6). It should be noted that downregulation of a given gene by insertion of an antisense sequence is likely to be more finely regulated, as opposed to overexpression. The antisense sequence technique does not include artificial production of any foreign protein in cells and therefore underlines the validity of the inhibitory capacity of RAP-2. However, it should be remembered that

the above low concentration jump is projected, not inverted, into the antisense half of the graph (Fig. 6).

To assess the diversity of transcription systems in which RAP-2 may participate, we have further studied c-Jun, a nuclear factor whose role in generating and maintaining an adequate response to stress is considered to be almost as critical as the role of NF-kb. Using the components of the commercial Path Detection System (Stratagene), we confirmed a similar biphasic effect of RAP-2 relative to several known AP-1 activators in HEK-293T and HeLa cells (see Figures 5B and 6B).

Example 5: RAP-2 potentiates c-Jun hyperphosphorylation without affecting JNK activity

To study the mechanisms of such significant effects on transcription, it is essential to determine the exact level at which normal signaling is disrupted. The transactivating potential of c-Jun is known to be regulated by the extracellular signal-induced phosphorylation of two serine residues ( ⁶³ Ser and ⁷³ Ser) at its amino-terminal activation domain. The JNK / SAPK protein kinases responsible for the above phosphorylation form a quite different subset of MAP kinases and are themselves activated by phosphorylation at Thr and Thr, which is mediated by other previous dual specificity kinases. Thus, the phosphorylation status of suitable sites in c-Jun and JNK can be used as a marker reflecting protein activation. Western blotting with lysates of transiently transfected HEK-293T cells showed that despite disruption of c-Jun mediated transcription, RAP-2 markedly increases endogenous c-Jun phosphorylation to ⁶³ Ser induced by various stimuli (see Fig. 7A). Total cell lysates of HEK-293T cells transfected with the indicated expression constructs together with either the pcDNA3-carrier labeled (-) in Fig. 7 or the pcRAP-2 labeled (+) in the same figure were analyzed on SDS-PAGE, were transferred onto the ECL-membrane and were probed anti-f osfo- ⁶³ Ser-c-Jun Ab (NEB). The membrane shown in the lower panel of Fig. 7A was re-probed with antibodies against total c-Jun for control (NEB).

The total amount of c-Jun remained - however - unchanged, except for elevation of c-Jun concentrations as a possible source of modification. Antibodies specific for the phosphorylated form

JNK1 / 2 did not detect any significant increase in the amount of these activated kinases in response to overexpression of RAP-2, suggesting that further c-Jun phosphorylation was not due to the enhancement of RAP-2-induced JNK activity (Fig. 7B). Activated

JNK1 / 2 from HEK-293T cells transfected with either pcDNA3 or pcRAP-2, treated for a prolonged period of time with hrTNFα, 183/185 were detected by western transfer of lysates with phospho- (Thr / Tyr) JNK Ab (NEB) as shown in FIG. 7.

In another experiment supporting the previous findings, an in vitro assay with immunoprecipitated JNK1 and purified GSDT-c-Jun as substrate produced essentially the same results (Fig. 7C). HEK-293T cells were co-transfected with the empty vector, pcRAP-2 and pcRIP in various combinations with each other together with the HA-JNK1 expressing plasmid. JNK1 was then immunoprecipitated via its N-terminal HA-terminal and its ability to phosphorylate bacterially produced purified GST-Jun was determined by incorporation of ³² P in an in vitro kinase assay. Reaction products were analyzed by SDS-PAGE as shown in Figure 7.

RAP-2 becomes phosphorylated when RAP-2-IKK1 complexes, immunoprecipitated from transfected HEK-293T cells, are incubated under phosphorylation conditions in vitro. The search for the functional role of RAP-2 phosphorylation showed that mutation of one lilac in this protein (at position 148) completely abrogates the activation of Jun RAP-2 phosphorylation. As shown in Figure 13, while overexpression of wild-type RAP-2 results in a significant increase in Jun phosphorylation, over-expression of RAP-2 (S148A) does not affect Jun phosphorylation.

However, the effect of RAP-2 on NF-κB is not affected by this mutation. These findings suggest that serine 148 RAP-2 is specifically involved in its effect on Jun phosphorylation.

Example 6: RAP-2 does not inhibit c-Jun and RelA binding to DNA

Since the experiments described in Example 5 did not reveal the cytosolic modulation target of RAP-2 overexpression in the NF-κB and AP-1 signaling cascades, we investigated the integrity of the nuclear processes necessary for transcription. The electromobility shift assay (EMSA) performed with nuclear extracts of transfected HEK293T cells unequivocally demonstrated that RAP-2 did not interfere with c.Jun and RelA binding to oligonucleotides corresponding to their classical recognition sequences (Fig. 8). In fact, a multiple enhancement in the efficiency of DNA / AP1 complex formation in RAP-2 transfected cells was observed. Furthermore, no interaction was observed between RAP-2 and c-Jun / RelA that could lead to steric obstruction of cJun / RelA activation domains. The effect of RAP-2 entry into the nucleus is expected to be at the level of events following binding of transcription enhancers.

Example 7: RAP-2 interacts in vivo with histone acetyltransferase TIP60

TIP60 (GeneBank U 74667) belongs to the newly described family of nuclear proteins called histone acetyltransferases (HAT). The enzymatic activity of these proteins is associated with the state of the chromatin structure in nucleosomal complexes.

HATs are often associated with some elements of the transcription machinery and are capable of affecting the rate of transcription. HATs cause chromatin relaxation near the initiation sites through the transfer of acetyl groups to specific histone lysine residues, allowing various factors access to DNA. They are one of those helper proteins that facilitate communication between the binding factors for enhancer and RNA polymerase II. Therefore, we investigated whether TIP60 can complex with RAP-2. Immunoprecipitation from HeLa cells, followed by a two-hybrid assay, showed that RAP-2 strongly interacts with TIP60 in both systems. However, we were unable to observe any significant alteration of RAP-2 mediated effects on NF-κΒ and c-Jun while co-expressing TIP60 in HEK-293T cells (not shown).

The same lack of alteration was observed in the control, i.e., stimulation ± TIP60 (w / o RAP-2), leading to the conclusion that a short reading time (20-30 hours post transfection) probably excludes the chance of reporter DNA to has become chromatinized, which does not provide enough time for the effect of HAT-like enzymes.

Example 8: Clone # 10 - New RAP-2 interacting proteins

Using the full-length RAP-2 protein as a capture reagent in the two-hybrid screening of the B-cell cDNA library, we isolated a new RAP-2 interacting protein designated as clone # 10 or protein encoded by clone # 10 or RAT-binding protein # 2. 10 or RBP-10 (FIG. 10). the original clone (approximately 2.2 kb) was found to encode a polypeptide of distinct molecular weight of 60 kDa. However, this sequence clearly lacks the putative ATG first codon. Despite the significant length, the obtained cDNA was therefore expanded further towards the 5 'end to reconstitute the entire open reading frame.

«· T t t t t · · · ·« «« · t t t t t t t t t t t t t t t ···· · ··· ··· ·· ··

The two-hybrid binding repertoire assay of clone # 10 showed that this protein has, in addition to RAP-2, a fairly strong affinity for TRAF2. However, clone # 10 does not bind to RIP, TRADD, MORT1,

MACH, TNFR-I, TIP60, and NIK, as well as several control proteins (e.g., laminin and cyclin). However, it cannot be excluded that the binding of clone # 10 to NIK can be detected in mammalian cells due to the peculiarities of the behavior of NIK in yeast. Clone # 10 has been shown to bind to RAP-2 in the C-terminal 200 amino acids of RAP-2, a region not necessarily associated with RIP, TIP60, NIK, and ββ binding.

Co-expression of clone # 10 with TRAF-2 in mammalian 293T cells prevented TRAF-2-mediated activation of NF-κΒ, while co-expression of clone # 10 with NIK strongly enhanced NF-κΒ NIK activation. these findings suggest a significant regulatory function of clone # 10. The different modulation effects observed indicate the probable existence of different, non-overlapping sites of action of the protein on the cell.

Several rounds of GenBank searches to identify the homologue of clone # 10 led to the identification of F40F12.5 (Accession # S42834) - a hypothetical C. elegans protein whose physiological role is unknown. Interestingly, F40F12.5 shows some similarities to several members of the highly conserved ubiquitin-protease family. These enzymes neutralize the destructive effects of the ubiquitination apparatus, which is involved in most of the events associated with protein degradation in the cell. While ubiquitin ligases are responsible for binding the polyubiquitin chain to a protein to be degraded, ubiquitin proteases prevent efficient branching of the growing chain. However, the predicted function of F40F12.5 based on similarity to the above-mentioned ubiquitin-driven proteases is questionable and it has not yet been established whether this protein has 44 44 Any enzymatic activity with respect to ubiquitin polymers. In addition, several facts make such coincidence quite unlikely:

(a) residues believed to form the core of the catalytic region in any ubiquitin-protease subclass are not conserved in either F40F12.5 or RBP-10;

(b) with the exception of their catalytic sites, enzymes from the ubiquitin-driven protease family from different species (from bacteria to humans) show essentially no sequence similarity, whereas F40F12.5 and clone # 10 show some degree of homology.

Example 9: Clone # 84: RAP-2 interacting protein

Another binding protein was identified using the full-length RAP-2 protein as a capture agent in a two-hybrid search of the B-cell cDNA library and this protein was designated clone # 84.

Clone a.84 was found to specifically bind to complete RAP-2 and at the same time showed no interaction with any other protein analyzed including TRAF2, MORR1, TRADD, RIP, NIK, TIP60 and lamin. The partial 5 'sequence of clone # 84 was found to be identical to the sequence of a previously cloned cDNA encoding a CGR19 cell growth regulating protein that was identified as a transcript specifically overexpressed in cells containing the p53 protein (Madden, S. et al., 1996, U66469). Sequence analysis of CGR19 led to the identification of the C3HC4 zinc finger motif (also referred to as the RING finger) at its C-terminal domain. CGR19 expression was found to suppress the growth of several cell lines. The involvement of CGR19 protein in NF-κΒ regulation by binding to RAP-2 may indicate modulation of the cell cycle regulatory network by members of the TNF-R family.

Example 10: Structural-functional relationships of RAP-2

A. Binding regions

Through sequential deletion analysis, the binding regions in RAP-2 were located and binding sites for RIP, NIK, TIP60 as well as self-association domains were identified (Fig. 11).

Binding to RIP was localized to the RAP-2 region of the protein that begins between amino acids 177-218 and ends at amino acid 264.

To date, the binding sites for β (amino acids 95-264) or NIK (amino acids 1-264) have not been found to overlap with the RIP binding site in RAP-2 (Fig. 11).

Binding to TIP60 is localized to the region between amino acids 95-264. The lack of interaction of the deletion fragment spanning amino acids 95-309 is most likely due to the specific downstream conformation that is made possible by this deletion.

A similar mismatch in binding to the deletion fragments was noted for clone # 10 binding and for self-association of RAP-2. In contrast to TIP60, the fact that complete RAP-2 binds to the deletion fragment containing amino acids 218-416, as well as to the deletion fragment containing amino acids 1-264, indicates that the region involved in homodimerization is located between amino acids 217-264.

The protein encoded by clone # 10, with the above exceptions, binds in a region beginning between amino acids 218-309 and ending at amino acid 416, and so its binding site may contain overlapping regions with RIP binding sites,

NIK, β and TIP60 (Fig. 11).

B. Functional regions

To our knowledge, all functional effects of RAP-2 (specifically NF-κΒ inhibition and c-Jun hyperphosphorylation induction) are located in the same region (Fig. 11).

Furthermore, it is possible that the region sufficient to efficiently influence signaling by all inducers used in the experiments is located at the N-terminal segment of the protein.

The region consisting of amino acids 95-416 has an effect, although significantly weaker compared to that of the full-length protein and may therefore be due to enhanced aggregation of endogenous RAP-2.

Further, with the exception of RelA, the effects of all inducers used in our experiments can be mediated by only about 100 N-terminal amino acids of RAP-2. Even a fragment containing amino acids 1-102 mediates a distinct effect, albeit somewhat weaker (Fig. 12B).

On the other hand, successful induction of RelA requires a much longer portion of the RAP-2 protein. So far, we could define the boundaries of this region between amino acids 1 and 264, which contains a region between amino acids 157 and 264, which has certain specific binding properties associated with RelA.

C. Linkage Relationships - Functions • ♦ • 9 »* • 9 9 9

9

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From the results shown in Figures 11 and 12, it appears that:

(a) with the exception of RelA, binding of RAP-2 to RIP, clone # 10, and most likely to NIK and TIP60, is not required for protein function as an inhibitor of NF-κB-induced overexpression.

(b) The effect of RAP-2 on RelA overexpression induced activation appears to be mediated, at least in part, by various binding events. Essentially, all of the above proteins may contribute to said activity, as determined from experiments conducted to date.

The exact site of interaction between RAP-2 and RIP has yet to be determined as it appears to be specific for RAP-2 and RIP and is not common to other proteins known to interact with RIP, such as MORT -1, TRADD, FAS-R and possibly also TRAF2 (see Malinin et al., 1997). It also appears (from sequence analysis and comparison to sequences in different databases as mentioned above) that RAP-2 does not contain a lethal domain, a MORT module, a protease domain (e.g., ICE / CED3 motif), a kinase domain / motif, or a TRAF domain .

At the same time, biological activity analysis also showed that RAP-2 has the following characteristics:

(i) overexpressing RAP-2 strongly inhibits NF-κB activation by TNF or by overexpressing the TRADD, RIP, TRAF-2, NIK, or p65 NF-κB subunit;

(ii) RAP-2 potentiates c-Jun hyperphosphorylation without affecting JNK activity;

(iii) RAP-2, as shown by deletion analysis, does not require the RIP lethal domain or RIP kinase activity for binding to RIP;

(iv) according to the above deletion analysis, the binding region was

RAP-2 for binding to RIP located around the N-terminal region of about 200 amino acids;

(v) RAP-2 binds to NIK in transfected mammalian cells but not in yeast.

Accordingly, RAP-2 therefore appears to be a highly specific binding protein for RIP and thus a RIP mediator / modulator that is likely to participate in the RIP-mediated intracellular signal transduction pathway.

Accordingly, RAP-2 appears to be involved in influencing / mediating RIP activities. Intracellularly, RIP is involved in the cell survival pathway (NF-κB activation, probably through interaction with TRAF2), and in signaling pathways leading to inflammation and cell death (independent of its killing domain or through interaction with other proteins such as MORT) -1, TRADD, p55-R, FAS-R and associated proteases such as MACH, Mch4, G1 and the like). The likely ways in which RAP-2 can affect / mediate RIP activity are detailed above. For example, the RAP-2-RIP interaction may result in an enhancement of the cell death or cell survival pathway, or may result in an inhibition of the cell death or cell survival pathway, which enhancement or inhibition is likely to depend upon the relative activities of the other elements of the two opposing intracellular signal transduction pathways. RAP-2 can also act as a storage protein causing the aggregation of many RIP molecules and other RIP- and RAP-2-binding proteins, where these aggregates can act in both the cell death and survival direction (or both), depending on on the relative activities of the other elements of the two intracellular signal transduction pathways in the cell.

• · · 44 4 4 ♦ 4 4 ♦ # 4 4 4 4 4

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Example 11: Preparation of polyclonal antibodies to RAP-2

The rabbits were first injected subcutaneously with 5 μρ of pure RAP-2 emulsified in complete Freund's adjuvant. Three weeks later, another subcutaneous injection of 5 μς of RAP-2 in incomplete Freund's adjuvant was given. Two additional injections of RAP-2 solution in PBS were given at 10 days interval. Rabbits were bled 10 days after the last immunization. The development of antibody concentration was monitored by radioimmunoassay. I-labeled RAP-2 was mixed with various dilutions (1:50, 1: 500, 1: 5000 and 1: 50000) of rabbit serum. A protein-G agarose bead suspension (20 μΐ, Pharmacia) was added in a total volume of 200 μΐ. The mixture was allowed to stand for 1 hour at ambient temperature, then the beads were washed 3 times and the bound radioactivity was counted. Rabbit antiserum to human leptin was used as a negative control. The RAP-2 antiserum titer was measured compared to the negative control titer.

Example 12: Preparation of monoclonal antibodies to RAP-2

Female Balb / c mice (3 months of age) were first injected with 2 μρ of purified RAP-2 emulsion with complete Freund's adjuvant and three weeks later injected subcutaneously with incomplete Freund's adjuvant. Three additional injections were given at 10-day intervals, subcutaneously in PBS. The final loading dose was administered intraperitoneally 4 and 3 days prior to the fusion of the mouse that showed the highest binding titer in IRIA (see below). Fusion was performed using an NS0 / 1 myeloma cell line and lymphocytes prepared from both the spleen and lymph nodes of the animal. Fused cells were plated and hybridomas were selected in DMEM supplemented with HAT and 15% horse serum. Hybridomas that have been 99 · ♦ * 3 9 4

· Confirmed production of antibodies to RAP-2, were subcloned by the limiting dilution technique, and injected into Balb / c mice prepared for landing for ascites production. Isotype antibodies were defined using a commercially available ELISA kit (Amersham, UK).

Screening for hybridomas producing anti-RAP-2 monoclonal antibodies was performed as follows: hybridoma supernatants were tested for the presence of anti-RAP-2 antibodies by solid phase inverted radioimmunoassay (IRIA). ELISA plates (Dynatech Laboratories, Alexandria, VA) were coated with Talon-purified IL-18Bpa-His6 (10 gg / ml, 100 μΐ / well). After overnight incubation at 4 ° C, the plates were washed twice with PBS containing BSA (0.5% and Tween 20 (0.05%) and blocked in the wash solution for at least 2 hours at 37 ° C. Hybridoma culture supernatants were added. (100 gg / well) and the plates were incubated for 4 hours at 37 DEG C. The plates were washed 3 times and goat-anti-mouse antibody-horseradish peroxidase conjugate (HRP, Jackson Labs) was added for 2 hours at ambient temperature. , 1: 10000, 100 μΐ / well) Plates were washed 4 times and color developed using ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (Sigma) with H2O2 as substrate) Plates were read by an automatic ELISA plate reader Samples showing an OD of at least 5-fold higher than the OD of the negative control were considered positive.

RAP-2 antibodies can be used to purify RAP-2 by affinity chromatography.

Example 13: ELISA assay

Microtiter plates (Dynatech or Maxisorb, Nunc) were coated with anti-RAP-2 monoclonal antibody (serum-free hybridoma supernatant or ascites). immunoglobulin fluid) overnight at 4 ° C. Plates were washed with PBS containing BSA (0.5%) and Tween 20 (0.05%) and were blocked in the same solution for at least two hours at 37 ° C. Test samples were diluted in blocking solution and added to the wells (100 μΐ / well) for 4 hours at 37 ° C. Plates were then washed 3 times with PBS containing Tween 20 (0.05%), followed by addition of rabbit anti-RAP-2 serum (1: 1000, 100 μΐ / well) and further incubation overnight at 4 ° C. Plates were washed 3 times and goat-anti-mouse antibody-horseradish peroxidase conjugate (HRP, Jackson Labs, 1: 10000, 100 μΐ / well) was added at ambient temperature for 2 hours. Plates were washed 4 times and color was developed using ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (Sigma) with H 2 O 2 as substrate. Plates were read by an automatic ELISA plate reader.

Having fully described the invention, it will be apparent to those skilled in the art that the invention may be practiced within a wide range of equivalent parameters, concentrations and conditions, without departing from the spirit and scope of the present invention and without undue experimentation.

Although the invention has been described in terms of certain embodiments thereof, there are further modifications thereof. The invention encompasses any variations, uses, or adaptations that follow the principles of the invention and include those deviations from the present disclosure that may be due to conventional practice in the art to which the invention pertains and which correspond to the general features set forth in the following claims.

All citations, including scientific articles or abstracts, 9 9 9 9 9 9 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 · 9 9 · · nebo nebo nebo nebo nebo nebo nebo nebo nebo nebo or other patent applications issued to U.S. Pat. or other patents or any other citations, are hereby incorporated by reference in their entirety, including all data, tables, figures and text of said citations. Furthermore, the entire contents of the citations cited in the citations of the present invention are also incorporated herein by reference.

References to known techniques, conventional techniques, known technique steps, or conventional method steps do not in any way include any aspect, description or embodiment of the present invention.

Thus, the description of specific embodiments fully describes the general nature of the invention that others, using the knowledge in the art (including references cited herein), can readily modify and / or modify the invention for various applications of such specific embodiments, without undue experimentation, without departing from the general of the concept of the present invention.

Therefore, such adaptations and modifications are within the meaning and scope of the equivalents of the disclosed embodiments. It is to be understood that the phraseology and terminology used herein is for the purpose of description and not of limitation and should be interpreted by a person skilled in the art in connection with that description in combination with the knowledge in the art.

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Sequence Listing <110> Wallach, David

Kovalenko, Andrei

Yeda Research and Devolopment Co., Ltd

<120> TGF / NGF receptor family modulators and other proteins <130> TNF / NGF receptors <14 0>

<141>

<150> 123758 <151> 1998-03-19 <150> 126024 <151> 1998-09-01 <160> 3 <170> PatentIn Ver. 2.0 <210> 1 <211> 2009 <212> DNA <213> Homo sapiens

CSuCígolCCc ttcggagcgc cacggactct ccgccLcaga gagtctccrc GLgtgggcct cggcccccta gctgacaccc cggtgcagcc tggcgaagcc gsgaggccca ccagcgttca c:; cccctct caccgctggc agccacgccg gcaaccgcgg cag ~ ccgccg tggacgaaca ccggcagcag caccěgccc ' accgcgaaac ctcccacccc C o u r C · - s_ r. and tcaggaccc tgcgaccttt tctcacgrct gaagaaccaa actgggcgaa cgctcctgag 60 120 2 4 0 ť Λ Τ ' gCCCČCCgGC gccgcctgga ggagaatcaa gaccccccag a tc c and tcc g gcagagcaac c c cagatcccgc gggagcgctg cgaggagccc ctgcaccccc a and g c c and g c c a aagggaggag / 'Yeah aaggagtccc ccacgcgeaa gctccaggag gccaggaaac tggtggagag actcggccrg / c o. cacaaccccc a t c t ga and g and g ccacaaccac cacgcc cc ~ c cccaccccca gcacctcaag Z • -id - · * ”· --- ¹ ~ agacgccagc agcagacggc cgagcacaag ccctccgcga aagcccaggc gacgtccttc 600 cccggggagc tgcaggagac ccagagccgc "" Gg ^ gcc_g ccaccaagca atgccaggct 6 50 ccggaggccc gggcccgggc ggccaacgag caggcgcggc a c c c c g and c a g tgagcgcgag 7 20

gcgctacagc agcagcacac cgtgcaggtg gaccagccgc gcacgcaggg ccagagcgtg caggccgcgc tccccatgga gcgccaggcc gccrcggagg agaagaggaa gctggcccag ttgcacctgg cccaccacca cctcttccaa gaataccaca ac ca ca ca a cagcaccg-g c-gggcagtc agcggaagcg accaacocac ccggaagarc L-Caaacagca actccagcag gccgaggagg ccccggcgcc caaacaggag gtgatcgata agccgaagga gcaggccgag cagcacaaga tcgtgacgga gaccgtcccg gtgctgaagc cccaagcgga tatctacaag

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gcggactccc aggccgacag gcaggcccgg caggagcagc tggaccagcc gcagagggag tcggccagca tcgaggacat gaggaagcgg cccacccccg cctacctctc CLCLCCCCtg gaggagccac ctgacccctg etgtcccaag ctgcagatac atgccat gga gcgcattgag ccgsggsccz ccccgggacc gcgcagtctg atgaagggc- gggtggccac aaccgggatg cggcacccca ege cc ca g c— gttaattccg ccgatcacgc ctgactcgcč gctctctttg cgagcraacc CCtCCCtCuC cctccacccg gccctcaggg agaacCgccc cccctggcag CaCCCaCCCg cccgctgccg rgccctggga tetcctttcg ggccgcatgc tattccattt · cactattgac ggacacctcc gttgtttccc akaaaaatcc tcg — gcacca 53585.3353

gagaagctgc ccgagaagaa ggacctcctg 114 0 tacagcaaac cgaaggccag ctgtcaggag 1200 catgtcgagg ccccccaggc ccccttgccc 1260 gccctgccca gccagaggag gagccccccc 1320 tgccagtatc aggcccctca tatggacacc 1380 tagggccgqc cagtgcaagg ccactgcc ^ g 144 0 cgctttcctc ccccgcctgc ctagcccaga 1500 ccacctggag ccccacccag gagctggccg 1560 ctggtcccct cctctggggc agatgcggcc 1620 ttcccttctg tccgctcgaa ccacttgcct 1680 gcactgggga agccaagaac ggggcctggg 1740 agctgggtgg cagctcttcc t cccaccgaa 1800 gtgctgccct cccaccatgc acaccggtgc 1860 tgcagccaga ccgatgtgta tttaaccagt 1920 atctttttgt caccatrnaac artggcmtag 198 0 2009

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tcctactcct cccccccccc cactccgcgc ccagtccaga gacacacccc gttcsccaaa tctccggaaa tgcctcacac aLag ~ tzgac cacctccgga agagccaacc gtgtcsgatg caggacgcac cgggcgaaga gtctcctctg gaacagggcg ctcetgagae zzzczagzaz cgccacccgg cagcagcaac cagattcttg ÍÍCC33GCC8 gccagaggga ggacaacgag aaaccggcgc agagaccccg cctggagaag ccgcgggagg cggagcaccc caagacatgc gtgaaageee aggtgacgcc cttgctcgcg gccgccacca aggaacgcca gcctctggag cggcagcccg agagcgagcg cgaggcgctg ccgcecacec agggceagag cgtggaggcc gaggagaaga ggaagccggc ccagttgcag gaeaaeeaea ccaagagcag zgzggzgggc gaceccaaac agcagcccca gcaggccgag gataagetea aggaggagge cgagcagcac aagccecagg cggacaccca caaggcgcac

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tccgacccca ccccctgcgt gagcactcc <. 60 cttgaccgcc ccctaccgag crtccttsss l. 12 0 agccagcccc cgccctgttg gazgaataag 180 gcgcagceea ccggtggccc ggeageaga 24 0 gggaagccag ccatgctgca cctcccttcs 300 tgcccgggac cacaatcaag acccccgaga 360 cgggagctgc egaagggagc tttctgcatt 420 ucccccaccc ccaagtc cca ggaggccagg 480 cccgacccga agaggeacaa ggagcaggct 540 cagcagcaca cggctgagga caaggcctcu 600 gagccgcagc agagccacag tcccttcgag 660 ggtcgcgccc ccgcggccag cgagcaggcg 720 cagcaccacc acagcgtgca cgtggaccag 780 gcgccccgca cggaccgcca ccccccctcc 840 gcggcccacc accagccccc ccaagaatac 900 agtgagcgea ccacctccaa 960 gaggcccccc aggccaaaca gcaggtgatc 1020 aagaccgcca tccagaccct tccggtgctg 1080 ccccagcccc agaggcaggc ccgggagaag 114 0 cacc-gcacc acccgcacoxg ccactacacc 1200 aggacccagc acacgaggaa gccccatgtc 12 60 cctgcccacc cccccccccc cccggccctg 1320 ccacccgacc cccgcccccc caagtgccaa 138 0 atacacgcca cggagcgcac tgactaggcc 1440 acgcgcccgg caccgcgcag tctgcgcttt 1500

100 ALIGN!

cctctcccgc ctgcctagcc caggatgaag qqczqqqzqa ccacaacccg ·· · aargccacct 15 6V ggagccccac ccaggagctg cccgcggcac cttacgctcc agctgtccat tccgctggtc 1620 ccctcttttg gggtagatgc caccccgatc aagcccgact cgctgctcct t gttcccfc 1680 tctgtctgct cgaaccactt gcctcgggct aatccctccc ccccccccca cccggcactg 1740 aggaagtcaa gastggggcc cggggcuccc agggaaaact gctccccccg gcagagctag 1800 gtggcagctc tíCCÍCCCcC cggacaccga cccgcccgct gccgcgcccc gggagtgctg 1860 ccctcttacc atgcacacgg gtgccctcct tttgggctgc acgccatccc aaattgcagc 1920 cagaccgatg c gc auccaac cagtcactac tgatggacat ctgggttctt tcccatcttt 1980 ttattaccat maatarcggc mcagakaaaa atccttgcgc actaaaaaaa and a and a 2034

9 • ·

9 9 9 <210> 3 <211> 2116 <212> DNA <213> Homo sapiens <400> 3 gccacgaagg cccagaccct gaccgtcctt cactgaccac

gctgaagtta ggactgaatg ggaacctcca cccaacacct gctggatcgg tcagccacc agtgtgcagg ccgtacgga agaaggcgcc gcttgtgaa agccggtctc caatcaagac tgagcgccgc aa gaagcagcga agaaaacacc ccaccaaaaa gggaaagaag aaaggcaccc tetkgcttat tckgccccta aaaaagaaac gacgccagaa ctaatcctcc gagaacacac tacctgaaaa ggcggaggcc tctcgaatac cecgccccac caggccaaaa cgcacaaeac ttggcgttcc cacaacccag ccgcagagcc accaccacgc cacccaaaaa acc-CCCCCC agacagcgcc ggacacgcgg cggacaccag ccggaaaaac acccgaacac ccccaaccac cgggagatcg gagacacgg ·: gcacagaaac aagccacca tcccccccgg acagcaccc: agccmcccmc gscccagaa · wcyccctcca cccccagga; acacgcgcca cgcacccagí gaaaggcaaa eaaaecegac ccccgcccac g gc cc accgc aacccccccc ctcaaaaacc aagggccact gccccgtccc CACC WC w KWG cgacacg uc c gcaccaggac cacaccccaa cgccacccct cacccgccag ccgaccaccc ccccggaatc agggcccgca aageagetet aaacacaacc cgcacccccc gccgcccctg cgacccggga ~ c GGAG AGC aaccccaagg gcccaacaac gccagagtcc cggcaatgga aaccgcctct caccaccacc taccacccag ctccgccagc cctcggccac aggagaaccc aagcgcccgc gaggcacccg ccacgcccga cccccag tggaaaaaga acaacCcccg nggacaccgg mmacccaaga gcgccacaaa ttacccccga ggccagaacc accaaacccc aacggcctcc agacgccccg acacacaaca acc gca cagcgcc gccacaa tcaacca cc:

ccccgaagac ccgcgcacca gag ^ a-g-ac ctaacgrrgc

Zqzczzzqzq cczqcczccz

uc i ~ c <SCC <s <STG ccagtccgca gccccccggg teetttccac tggactgcaa gcacttcacc ccccaggccu cacttggagg argcccggag kcacccagac ccccacccca gccaccgagg aaccaccaaa acaaaaaga ~ cccgctaaaa cacggaaaaa caccaacagc acccggaaaa gacccacccg gcaagaacoc caacacccaa ccccaaagac ggagtcaccc caaccacgac aatggcccaa gccccctgga agaccgccc * _ aaacaaccgg ac cc CACC cg gcgacgaccc accacccaac cccgtgaact atgcccaaca acggaagagc aacccacacg ggggtaatcc ccggaagacg cgcgccccga ccaccaccgc caacttaagt acaacgactg tcaaccccac gaccccaaag a. cacaaacug

120 ISO 240 300 360 4 20 4 80 540 600 660 720 780

ccgaggaaaa 840 cccgaggaac 900 and L.cay if Ou aacgagaaag 1020 aacccgaaat 1080 gsctttaaac 114 0 aagacaccce 1200 taegaegate 12 60 gcccaccctc 1320 tc accccgac 1380 cccgctetet 1440 rcagcctggc 1500 caatccccca 1560 agaccctcsa 1620 gc gacgccat 1680 ggctcatcgg 1740 gagctggcag 1800 cccagaaaag 18 60 aagaagcacc 1920

44

4 4 4 5 6 7

44 ·

101 4 4 4 4 9 4 9 44 94 4 4 443 4 4 • 4 4 9 4 • • · írgcacccta G? 3gC | Ig-C ttgtgttggt tttttaagaa Gt Ct3Sa ~ G5 sge ~ a11 and 31 1980 acctgaagct tt35gtt.33G tgcattgatc atatgatatt rttggaagca u3C33lul33 2040 attatggaag tG ~ 3S3GCCG cttttagtcc attgagaatg LcSatSaSLg tgtcttctet 2100 5i 333333 2116

• 9

Claims (38)

Patent claims A DNA sequence encoding a RIP-associated protein (RAP-2), isoforms, fragments or analogs thereof, wherein said RAP-2, isoforms, fragments or analogs thereof are capable of binding to RIP. DNA sequence according to claim 1 encoding RAP-2, isoforms, fragments or analogs thereof which are capable of affecting or mediating the intracellular activity of RIP. A DNA sequence according to claim 1 or 2 selected from the group consisting of: (a) DNA sequences derived from the natural region coding region RAP-2 protein; (b) DNA sequences capable of hybridizing to sequence (a) under moderately stringent conditions that encode biologically active RAP-2 protein; and (c) DNA sequences that are degenerate due to the degeneracy of the genetic code relative to the sequences defined in (a) and (b) that encode a biologically active RAP-2 protein. DNA sequence according to any one of claims 1 to 3 comprising the sequence shown in Fig. 1. DNA sequence according to any one of claims 1 to 4 comprising the sequence shown in Fig. 2. 6. A DNA sequence according to claim 4 or 5 encoding a RAP-2 protein, isoform, analog or fragment thereof comprising at least a portion of the sequence depicted in FIG. 3. A replicable expression vehicle comprising a DNA sequence according to any one of claims 1-β, optionally operably linked to 99 β. r • control sequences, promoters, or other DNA sequences allowing expression in the correct orientation. The replicable expression vehicle of claim 7, which may be expressed in eukaryotic host cells. The replicable expression vehicle of claim 7, which may be expressed in prokaryotic host cells. Transformed eukaryotic or prokaryotic host cells comprising a replicable expression vehicle according to any one of claims 7-9. The RAP-2 protein, isoform, fragment, functional analogs or derivatives thereof encoded by the DNA sequence of any one of claims 1-6, wherein said RAP-2, its isoform, fragment, analogs or derivatives thereof is capable of binding to RIP. The RAP-2 protein of claim 11, which is capable of affecting or mediating intracellular activity of RIP in intracellular signal transduction pathways leading to inflammation, cell survival or cell death in which RIP participates directly or indirectly through association with other intracellular modulators or mediators of these tracks. The RAP-2 protein, isoform, fragment, analogs or derivatives thereof of claim 11, wherein said protein, isoform, fragment, functional analogs or derivatives thereof, comprising at least a portion of the amino acid sequence shown in Figure 3. A method for producing a RAP-2 protein, isoform, fragment, analogs or derivative thereof according to any one of claims 11 to 13, comprising culturing 104 ················································ Transformed host cells according to claim 9 under conditions suitable for the expression of said protein, isoform, analog, fragment or derivative, carrying out the post-translational modifications necessary to obtain said protein, isoform, an analog, fragment or derivative and isolating said protein, isoform, analog, fragment or derivative. Antibodies or active fragments thereof specific for the RAP-2 protein, isoform, fragment analog or derivative according to any one of claims 11 to 13. 16. A method for affecting or mediating intracellular events affected / mediated by RIP in intracellular signal transduction pathways leading to inflammation, cell survival or cell death in which RIP participates directly or indirectly through other intracellular modulators or mediators of these pathways, characterized in that: comprising treating said cells with one or more RAP-2 proteins, isoforms, analogs, fragments or derivatives of any one of claims 11 to 13 that are capable of binding to RIP and affecting or mediating said intracellular RIP activity, wherein said treating cells comprises introducing one or more a plurality of proteins, isoforms, analogs, fragments or derivatives into said cells in a form suitable for their intracellular transfer, or insertion of a DNA sequence encoding one or more proteins, isoforms, analogs, fragments or derivatives into said cells in the form of a suitable vector comprising said sequence, wherein said vector is capable of inserting said sequence into said cells in such a way that said sequence is expressed in said cells. 17. Method for influencing RIP affected / mediated 105. a cell effect according to claim 16, wherein said treatment of cells comprises inserting a DNA sequence encoding said RAP-2 protein, isoform, analog, fragment or derivative into said cells in the form of a suitable vector comprising said sequence, wherein said vector is capable of inserting said sequence into said cells in such a way that said sequence is expressed in said cells. The method of claim 16 or 17, wherein said treating cells is transfecting said cells with a recombinant animal viral vector, comprising the steps of: (a) constructing a recombinant animal viral vector carrying a sequence encoding a viral surface protein (ligand) capable of binding to a. a specific cell surface receptor on the surface of cells carrying FAS-R or p55-R and a second sequence encoding a protein selected from the RAP-2 protein, isoforms, analogs, fragments and derivatives thereof according to any one of claims 11 to 13 which is expressed in said cells able to influence / mediate RIP activities; and (b) infecting said cells with said vector of step (a). 19. A method for affecting a RIP affected / mediated effect on a cell, comprising treating said cells with antibodies or active fragments thereof according to claim 15, wherein said treating comprises applying a suitable composition comprising said antibodies, active fragments or derivatives thereof to said cells. wherein when the RAP-2 protein or parts thereof is present on the extracellular surface of said cells, said composition is ready for extracellular administration, and 106 wherein said RAP-2 proteins are intracellular, such that said composition is ready for intracellular administration. 20. A method for affecting RIP affected / mediated effects on a cell comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence for at least a portion of the DNA sequence encoding the RAP-2 protein of any one of claims 1 to 6, wherein said oligonucleotide sequence is capable of block the expression of RAP-2 protein. The method of claim 20, wherein said oligonucleotide sequence is inserted into said cells by the virus of claim 18, wherein said second sequence of said virus encodes said oligonucleotide sequence. 22. A method for the treatment of HIV-infected cells or other cells affected by the disease, comprising: (a) constructing a recombinant animal viral vector carrying a sequence encoding a viral surface protein capable of binding to a specific cell surface receptor on the surface of tumor cells or to a specific surface receptor on the surface of HIV infected cells or to a receptor on other cells affected by the disease; selected from the RAP-2 protein, isoforms, analogs, fragments and derivatives thereof according to any one of claims 11 to 13 which, upon expression in up to said tumor cells, HIV-infected cells or other cells affected by the disease, is capable of enhancing cell killing directly or indirectly RIP-mediated; and (b) infecting said tumor cells or cells 4 9 4 92 9 99 4 4 107 infected with HIV or cells affected by another disease of the vector of step (a); 23. A method for affecting RIP effects on cells comprising the use of a ribozyme technique, wherein the vector encoding the ribozyme sequence capable of interacting with the cellular mRNA sequence encoding the RAP-2 protein of any one of claims 11 to 13 is inserted into said cells in the form of allowing expression of said ribozyme sequence in said cells, and wherein said ribozyme sequence upon expression in said cells interacts with said cellular mRNA sequence and cleaves said mRNA sequence resulting in inhibition of overexpression of said RAP-2 protein in said cells. The method of any one of claims 16 to 23, wherein said protein is at least one of RAP-2 isoforms, analogs, fragments and derivatives. A pharmaceutical composition for influencing the effects of RIP on a cell, comprising as an active ingredient at least one RAP-2 protein according to any one of claims 11 and 13, a biologically active fragment, analog, derivative or mixture of said substances. 26. A pharmaceutical composition for influencing the effects of RIP on a cell comprising, as an active ingredient, a recombinant animal viral vector encoding a protein capable of binding to a cell surface receptor and encoding at least one RAP-2 protein, isoform, active fragments, analogs according to any one of claims 11 to 13. 108 ··· ··························· ··· ·· ·· 27. A pharmaceutical composition for influencing the effects of RIP on a cell comprising as an active ingredient an oligonucleotide sequence encoding an antisense sequence to the DNA sequence for the RAP-2 protein of any one of claims 1 to 6. A method for influencing processes affected / mediated directly or indirectly by RIP, comprising treating said cells with one or more RAP-2 proteins, isoforms, analogs, fragments or derivatives according to any one of claims 11 to 13 which are capable of binding to RIP, wherein said treatment of cells comprises introducing one or more proteins, isoforms, analogs, fragments or derivatives into said cells in a form suitable for their intracellular transfer, or inserting a DNA sequence encoding one or more proteins, isoforms, analogs, fragments or derivatives into said cells. cells in the form of a suitable vector comprising said sequence, wherein said vector is capable of inserting said sequence into said cells in such a way that said sequence is expressed in said cells. 29. A method for affecting processes influenced / mediated directly or indirectly by RIP, comprising inhibiting NF-κ J and activating JNK and p38 kinase, comprising treating said cells with one or more RAP-2 proteins, isoforms, analogs, fragments or derivatives according to any one of claims 11 to 13, which are capable of binding to RIP, wherein said treatment of cells comprises inserting one or more proteins, isoforms, analogs, fragments or derivatives into said cells in a form suitable for their intracellular transfer, or inserting a DNA sequence encoding one or a plurality of proteins, isoforms, analogs, fragments or derivatives into said cells in the form of a suitable vector comprising: 1 2 3 4 5 6 9 109 of said sequence, wherein said vector is capable of inserting said sequence into said cells in such a way that said sequence is expressed in said cells. A fragment according to any one of claims 11 to 13, which is a peptide. 31. A DNA sequence encoding a RAP-2 binding protein, isoforms, fragments or analogs thereof, wherein said RAP-2 binding protein, isoforms, fragments or analogs thereof are capable of binding to RAP-2. 32. The DNA sequence of claim 32 which encodes a protein capable of affecting / mediating RAP-2 function. 33. The DNA sequence of claims 32 or 33, which comprises the sequence shown in FIG. 10. A RAP-2 binding protein capable of binding to RAP-2 and / or affecting / mediating RAP-2 function. 35. A method for isolating and identifying proteins capable of binding to RAP-2, comprising the use of a yeast two-hybrid technique, wherein the sequence encoding said RAP-2 is contained in a single hybrid vector and the sequence from a cDNA library or genomic DNA library is contained in a second hybrid vector, wherein the vectors are used to transform a yeast host and the positive transformants are isolated, and then extracting said second hybrid vector to obtain a sequence encoding a protein that binds to RAP-2. 36. A RAP-2 binding protein which is a protein encoded by it 110 • Clone 10. 37. A method for affecting / mediating RAP-2 function comprising treating cells with one or more RAP-2 binding proteins. 38. The method of claim 37, wherein the RAP-2 binding proteins are selected from the group consisting of a protein encoded by clone 10 and CGR 19.