CZ20003098A3 - Proteins interacting with caspase-8 - Google Patents

Abstract

Caspase-8 interacting proteins, their production and use such proteins.

Description

Technical field

The invention relates to cysteine proteases. Proteins that interact with caspase-8 (MACH) and / or modulate its function in CD95 (Fas / Apo-1) TNF mediated death are described.

cells (or apoptosis) or CD120a (p55 receptor), in particular, proteins that interact with caspase-8 (MACH directly or indirectly) are also described. The invention also relates to the preparation and use of caspase-8 / MACH interacting proteins.

BACKGROUND OF THE INVENTION (described by Ion Gresser,

Tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) (TNF stands for both TNF-alpha and TNF-beta in this document) are more functional pro-inflammatory cytokines that are mainly mononuclear phagocytes that affect cells in Wallach, D (1986), Interferon 7 ed., Pp. 83-122, Academic Press, London, and Beutler and Cerami (1987)). Both TNF-alpha and TNF-beta initiate their effects by binding specific cell surface receptors. Some of these effects are likely to be beneficial to the organism: for example, they can destroy tumor cells or virus-infected cells and increase the antibacterial activity of granulocytes. In this way, TNFs are involved in the defense of the body against cancer and infectious agents, and are involved in post-injury recovery. TNFs can be used as anti-tumor agents that bind to receptors on the surface of tumor cells and thus initiate events leading to tumor cell death. TNFs can also be used as anti-infection agents.

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TNF-alpha and TNF-beta also have deleterious effects. There is evidence that overproduction of TNF-alpha may be the main reason for some serious diseases. For example, it is known that the effects of TNF-alpha, primarily on the vascular system, cause septic shock (Tracey et al., 1986). In some diseases, TNF can cause large weight loss (cachexia) by suppressing adipocyte activity and causing anorexia. TNF-alpha can therefore be called cachectin. It is also described as a mediator of tissue damage in rheumatic diseases (Beutler and Cerami et al., 1987) and as a major mediator of the damage observed in graft versus host reactions (Piquet et al., 1987). In addition, TNF is known to be involved in the inflammatory process and in many other diseases.

Two different independently expressing p55 (CD120a) and p75 (CD120b) TNF-R receptors that bind specifically to TNF-alpha and TNF-beta initiate and / or mediate the above-described biological effects of TNF. These two receptors exhibit structurally different intracellular regions, suggesting that their signals are different (Hohmann et al., 1989, Engelman et al., 1990, Brockhaus et al., 1990, Loetscher et al., 1990, Schall et al., 1990, Nophar et al., 1990, Smith et al., 1990, and Holler et al., 1990). The cellular mechanisms of the various proteins and probably other factors involved in the intracellular signaling of CD120a and CD120b have yet to be elucidated. These are intracellular signals that typically occur upon binding of ligands, i.e., TNF (alpha and beta) to the receptor, which is responsible for initiating a cascade of responses that lead to the observed response of the cell to TNF.

Regarding the above-mentioned cytocidal effect of TNF, which was studied in most cells, this effect is mainly due to CD120a. Antibodies against the extracellular region (ligand binding region) of CD120a can itself cause a cytocidal effect (see

See EP 412486), which correlates with the efficiency of cross-linking of antibodies to the receptor. This phenomenon is believed to be the first step in the formation of an intracellular signaling process. Mutation studies (Brakebush et al, 1992, Tarraglia et al., 19993) have shown that the biological function of CD120a depends on the integrity of its intracellular region, and it is also suggested that the initiation of intracellular signals leads to the cytocidal effect of TNF as a result of joining two or more intracellular CD120a regions. TNF (alpha and beta) appear as homotrimers, suggesting that, as such, they induce intracellular signals via CD120 through its ability to bind and form cross-links with receptor molecules. That is, they cause receptor aggregation.

Another member of the TNF / NGF receptor superfamily is the FAS / APO1 receptor (CD95), which is also called the FAS antigen. It is a cell surface protein expressed in various tissues and sharing homology with a number of cell surface receptors, including TNF-R and NGF-R. CD95 mediates cell death in the form of apoptosis (Itoh et al., 1991) and appears to serve as a negative selector of autoreactive T cells, i.e. at the time of T cell maturation. CD95 mediates opoptosis death of T cells that recognize self antigens. It has also been found that mutation of the CD95 (1pr) gene also causes a lymphoproliferative disorder in mice that causes autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al., 1992). A CD95 ligand appears to be a cell surface-associated molecule carried, inter alia, by deadly T cells (or cytotoxic lymphocytes-CTLs) and therefore, when such CTLs come into contact with CD95-bearing cells, they are able to induce apoptosis cell death carrying CD95. Monoclonal antibodies were further prepared to be specific for CD95. These monoclonal antibodies are capable of inducing opoptosis death of cells bearing CD95, including cells transformed with cDNA encoding human CD95 (Itoh et al., 1991).

Some cytotoxic effects of lymphocytes were mediated by the interaction of the ligand produced by lymphocytes with the widely occurring cell surface receptor CD95, which has the ability to cause cell death. They found that besides T lymphocytes, various other normal cells express the CD95 receptor on their surface. These cells can be killed by triggering this receptor. Such uncontrolled induction of the killing process is expected to contribute to tissue damage in certain diseases, such as liver cell damage in acute hepatitis. Thus, finding ways to suppress the cytotoxic activity of CD95 receptors may have therapeutic potential.

Conversely, it has also been found that certain malignant and HIV-infected cells carry the CD95 receptor on their surface, then antibodies against CD95 receptors or CD95 receptor ligand can suppress CD95 receptor-mediated cytotoxic effects in these cells. This makes it possible to combat malignant and HIV-infected cells (Itoh et al., 1991). Finding other methods for enhancing the cytotoxic activity of CD95 receptors may therefore have therapeutic potential.

A method for modulating the cellular response to TNF (alpha and beta) and CD95 receptor ligand is needed. For example, in the pathological situation considered above where TNF or the CD95 receptor is overexpressed, it is necessary to inhibit the cytocidal effects induced by TNF or the CD95 receptor ligand. In another situation, for example in wound healing, the effect of TNF must be increased or, in the case of CD95 receptors, in tumor cells or HIV-infected cells, the effect mediated by CD95 must be enhanced.

A number of approaches have been proposed (for example, in European patent applications EP 186833, EP 308378, EP 398327 and EP 412486) to regulate the adverse effects of TNF by inhibiting TNF binding to its receptor using anti-TNF antibodies or using soluble TNF receptors. essentially soluble extracellular regions of the receptors) to compete with TNF-binding for cell surface-bound TNF-R. Based on the finding that TNF binding is required for TNF-induced cellular effects (see, for example, EP 568,925), an approach has been developed to adjust the effect of TNF by modulating TNF-R activity.

For example, EP 568925 discloses a method for modulating TNF-R signaling and / or cleavage, wherein peptides or other molecules can react with either the receptor itself or with effector proteins that interact with the receptor to modulate the normal function of TNF-R. EP 568925 describes the construction and characterization of various mutant forms of CD120a having mutations in their extracellular, transmembrane and intracellular regions. In this way, CD120a regions that are essential for receptor function have been identified in the previously mentioned regions. That is, they are essential for ligand binding (TNF) and subsequent signal transduction and intracellular signals that lead to the observed effect of TNF on cells. In addition, a number of approaches to isolate and identify proteins, peptides or other factors that are capable of binding to different regions in the above CD120a regions are described, which proteins, peptides, and other factors may be involved in regulating or modulating TNF activity. -R. EPO 368925 also describes a number of approaches for isolating and cloning DNA sequences that encode such proteins and peptides for

Construction of expression vectors suitable for the production of these proteins and peptides and for the preparation of antibodies or fragments thereof that react with CD120a or the aforementioned proteins and peptides that bind to different regions of CD120a. EP 568925 does not specify actual proteins and peptides that bind to the intracellular region of TNF-R (for example the CD95 receptor), nor does it describe a yeast two-hybrid approach to isolate and identify proteins or peptides that bind to the intracellular region of TNF-R. Similarly, EP 568925 does not disclose proteins or peptides capable of binding to the intracellular region of CD95.

Thus, when it is necessary to inhibit the effect of TNF or CD95 receptor ligand, it is necessary to reduce the amount of TNF-R or CD95 receptor activity on the cell surface, while increasing the amount or reactivity of TNF-R or CD95 receptor will be required if enhancement of TNF is considered. or CD95 receptor. The CD120a and CD120b promoters were analyzed and sequenced to determine a number of key sequence motifs that are specific to various transcriptional regulatory factors, and thus the expression of these TNF-Rs can be controlled at the promoter level. This means inhibiting transcription from promoters to reduce the number of receptors and enhancing transcription from promoters to increase the number of receptors (EP 606869 and WO 9531206).

While tumor necrosis factor (TNF) receptors and the structurally related CD95 receptor are known to trigger destructive activities in cells upon stimulation with ligands produced by leukocytes that lead to their own destruction, the mechanisms of this trigger are still unknown. Mutation studies indicate that at the CD95 and CD120a receptors, the cytotoxicase signals include different regions in their intracellular regions (Brakebush et al., 1992, Tartaglia et al., 1993, Itoh and Nagata, 1993).

These regions (so-called dead domains) show sequence similarity). “The dead domains of the CD95 and CD120a receptors tend to associate themselves. Their association promotes the aggression of the receptors, which is necessary for the initiation of the signals (Song et al., 1994, Wallach et al., 1994, Bolding et al., 1995), and can lead to signals that are not ligand-dependent methods (Boldin et al., 1995).

Some of the cytotoxic effects of lymphocytes are mediated by the interaction of the ligand produced by lymphocytes CD95, an extended cell surface receptor that has the ability to cause cell death (Nagata and Goldstein, 1995) and the fact that mononuclear phagocyte killing involves ligand-receptor pairing TNF and its CD120a receptor, which is structurally related to CD95 and its ligand (see also Vandenabeele et al., 1995). Like other receptor-induced effects, the induction of cell death by TNF and CD95 receptors is manifested by a variety of protein-protein interactions, ranging from ligand binding to the receptor to eventual activation of enzymatic effector functions that induce non-enzymatic protein-protein interactions that initiate cell death signals. They are the binding of the TNF trimer molecule or the CD95 ligand molecule to receptors, with the resulting intercellular domain interactions (Brakebusch et al., 1992, Tartaglia et al., 1993, Itoh and Nagata, 1993) increasing the susceptibility of death region motifs. to self-link (Boldin et al., 1995a) and to induce the binding of two cytoplasmic proteins (which can bind to each other) to the MORT-1 (or FADD) intracellular receptor region with CD95 (Boldin et al. , 1995b, Chinnaiyan et al., 1995, Kischkel et al., 1995) and TRADD with CD120a (Hsu et al., 1995, Hsu et al., 1996). Three proteins that bind to the intracellular domain of the CD95 and CD120a receptors in the "lethal domain that is involved in the induction of cell death" using receptors through heteroasociation of homologous regions and which are also independently capable of causing cell death will be identified by a yeast two-hybrid screening procedure. one of these proteins is the MORT-1 protein (Boldin et al., 1995b), also known as FADD (Chinnaiyan et al., 1995), which binds specifically to the CD95 receptor. The second TRADD protein (also described in Hsu et al., 1995, 1996) binds to the CD120a receptor, and the third RIP protein (Stanger et al., 1995) binds to both CD95 and CD120a receptors. In addition to binding these proteins to the CD95 and CD120a receptors, they are also able to bind to each other, allowing functional signal penetration between the CD95 and CD120a receptors. Such binding occurs through the conservative sequence motif of the "death domain module" common to receptors and their associated proteins. Furthermore, although the yeast two-hybrid assay has shown that the MORT-1 protein spontaneously binds to the CD95 receptor in mammalian cells, this binding only occurs after receptor stimulation, suggesting that the MORT-1 protein is involved in the initiation of CD95 receptor signals. MORT-1 does not contain any sequence characteristic of enzymatic activity and therefore its ability to cause cell death does not involve the intrinsic activity of MORT-1 itself, but rather the activation of some other protein or proteins that bind to MORT-1 and act further in the signaling cascade. Cell expression of MORT-1 mutants lacking the N-terminal portion of the molecule has been shown to block induction of cytotoxicity by the CD95 or CD120a receptor (Hsu et al., 1996, Chinnaiyan et al., 1996), indicating that this N-terminal the region transmits signals for the cytocidal effects of both receptors through protein-protein interactions.

Certain studies have implicated a group of cytoplasmic thiol proteases that are structurally related to proteases

CED3 Caenorhabditide elegans and mammalian enzyme altering the induction of various processes (see interleukin-1 physiological beta (ICE) until Kumar cell death, 1995 and Henkart, 1996) . There are some indications that proteases of this family may be involved in CD95 and TNF-R receptor-induced cellular cytotoxicity. Specific peptide protease inhibitors and proteins encoded by two viruses that block their function, namely the chicken pox protein crmA and the baculovirus p35 protein, have been found to protect cells from this cellular toxicity (Enari et al., 1995, Los et al., 1995, Tewari et al., 1995, Xue et al., 1995, Beidler et al., 1995). Rapid cleavage of certain specific cellular proteins, particularly mediated by CED3 / ICE family proteases, could be demonstrated in cells shortly after stimulation of CD95 or TNF-R receptors.

Currently, one such protease and its various isoforms (including inhibitors), known as MACH (now caspase-8), which is a MORT-1 binding protein and is used to modulate MORT-1 protein activity, have been isolated, cloned and characterized and can act independently of the MORT-1 protein. 932 as in the corresponding PCT Applicant. PCT / US96 / 10521 and Boldin et al. 1996) is also its possible use. In addition, another such protease and its various isoforms (including inhibitors) that have been designated Mch4 (also called caspase-10) has been isolated and characterized (Fernandes-Alemri et al., 1996, Srinivasula et al., 1996). Caspase-10 is also a MORT-1 binding protein that serves to modulate the activity of the MORT-1 protein and therefore probably the CD95 and 120a receptors, and which can also act independently of the MORT-1 protein. Caspase-10 is disclosed in the above publications.

It should also be noted that caspases-8 and 10, which have similar prodomains (Boldin et al., 1996, Muzio et al., Fernandes-Alnemri et al., 1996, Vincent and Dixit, 1997, interact through their prodomains with the MORT protein. These interactions are present in the non-terminal part of the MORT-1 protein via the "death region motif or" death effector region, DED, and occur as a duplicate in caspase-8 and 10 (Boldin et al., 1995b, Chinnalyan et al. , 1995).

Such cysteine aspartate as caspases are growing (the cysteine family has several common features.

proteases, now known specific proteases) proteases that they share

Most such caspases have been found to be involved in the initiation and execution of programmed cell death or apoptosis, while others appear to be involved in the production of pro-inflammatory cytokines (Nicholson DW et al., 1997, Salvesen GS et al., 1997, Cohen GM 1997). . They are synthesized as catalytically inactive precursors and are generally activated by cleavage at the position of the internal aspartate residue present in the internal region of the linkers. Caspase cleavage sites are defined by tetrapeptide sequences (X-X-X-D) and cleavage always occurs after aspartic acid. The resulting certain mature active caspases can process and activate their own as well as other inactive precursors (Fernandes-Alnemri T et al., 1996, Srinivasula SM et al., 1996).

Activation of the programmed cell death process is generally specific and involves the sequential processing of caspases called "powerful caspases that are down in the caspase cascade and" initiation caspases that are up the caspase cascade. The functional characteristics of the two caspase classes also reflect their structure. "Initiating caspases contain a longer pro-region compared to" powerful caspases (Salvesen G.S. et al., 1997, Cohen G.M. 1997). The long pro-region allows initiating or apical caspase to be activated by triggering the deadly receptors of the TNF receptor family. After ligand-induced trimerization •

For the death receptors, the initiating caspases are selected on the basis of their long pro-region to react with specific adapter molecules to form a death-inducing signaling complex (Cohen G.M. 1997, Kischkel F. C. et al. 1995). For example, caspase-8 / MACH and probably caspase-10, which contains two lethal effector domains (DED and FADD), are selected for receptor complex with FADD / MORT-1 adapter molecules, while for caspase-2, CRADD / RAIDD proteins and RIP (Nagata S. et al., Cell 1997, 88 (3): 355-65. MacFarlane M. et al., J. Biol. Chem. 1997, 272 (41): 2541-20, Ahmad M. et al., Cancer Res 1997, 57 (4): 615-9, Duan H. et al., Nature 1997, 385 (6611): 86-9). Based on the trimeric nature of the activated receptor complex, it is thought that at least two caspase molecules are at a small distance from each other, leading to their activation by autocatalytic processing (Yang X. et al., Mol. Cell 1998, 1 (2)). Muzio et al., J. Biol. Chem., 1998, 273 (5): 2926-30).

Caspases are synthesized as proenzymes that contain three major subunits, the N-terminal prodome and two subunits, which are sometimes separated by a linker peptide. Two subunits are termed "long subunit or subunit 1, which subunit contains an active enzymatic site, and" short subunit or subunit 2. In the case of full enzyme activation, the prodomain and two subdomains are cleaved. The two cleaved subunits form a heterodimer, the long domain being derived from the N-terminus and the short subunit from the C-terminal region of the caspase precursor.

the deduced three-dimensional structures of caspase-3 appear to release the C-terminal end of the long domain, as well as the N-terminus of the short subdomain, and the C-terminus of the short subunit approaches the N-terminus of the long subunit to generate a properly folded active enzyme (Rotonda et al. , Nat Structure Siol 1996: 3 (7): 619-25, Mittl et al., PR et al., J. Biol. Chem. 1997, 272 (10)

On • ·

6539-47, Srinivasula SM et al., J. Biol. Chem. 1998, 273

10107-11).

N-acetylglucosamine-6-phosphate deacetylase is an intracellular enzyme known to be involved in the intracellular glucosamine metabolism. A genomic DNA fragment containing Nacetylglucosamine-6-phosphate deacetylase was cloned from the chitinase-producing Vibrio cholerae (Yamano et al., Biosci Biotevhnol Biochem. 61, 1349-53, 1997).

The nagA gene encoding N-acetylglucosamine-6-phosphate deacetylase of E. coli is also known (Peri et al., January 1990, 68 (1) pp. 123-137). To date, human N-acetylglucosamine-6-phosphate deacetylase has not been reported to be cloned or sequenced.

SUMMARY OF THE INVENTION

The invention provides a protein reactive with caspase-8 or an isoform, allelic variant, fragment, functional analog, mutant or derivative thereof that is capable of interacting with subunit 1 and / or subunit 2 of caspase8.

The invention further provides N-acetylglucosamine-6-phosphate deacetylase or an isoform thereof, allele variant, fragment, functional analog, mutant or derivative thereof.

Further, the invention provides a protein which has the amino acid sequence shown in Figures or 6.

containing 2, 3, 5B

The invention also provides a protein comprising the amino acid sequence of the Tip-60 protein with excluded amino acids 94-145.

Further, the invention provides a protein comprising the amino acid sequence encoded by clones P27, P70, P79, L7, L12, M26, B4, B17, J40, B13, B37, B33, or P74 and spliced variants P16 and P43 thereof as described below. .

The invention further provides a protein as defined above that is cleaved in vitro or in vivo by caspase-8.

The invention further provides an isolated DNA sequence encoding a protein of the invention. The present invention provides the DNA sequence shown in Figures 2 and 3. The invention further provides isolated DNA capable of hybridizing to said DNA sequence under moderately stringent conditions.

The invention further provides a vector comprising a DNA sequence as defined above.

The invention further provides a eukaryotic or prokaryotic host cell comprising a vector of the invention.

The invention further provides a method for producing a protein of its isoform, allele variant, fragment, functional analog, mutant or derivative of a caspase-8 interacting protein of the invention. The method comprises culturing a host cell of the invention under conditions that allow the production of said protein, post-translational processing necessary to obtain said protein, its isoform, allele variant, fragment, functional analog or mutant or derivative, isolating said protein of its isoform , allele variants, fragments, functional analogs, or mutants or derivatives.

The invention further provides said method, wherein the cell is a prokaryotic cell.

The invention further provides said method, wherein the cell is a eukaryotic cell.

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The invention describes said method, wherein the cell is a mammalian, insect or yeast cell.

The invention describes a method wherein the cell is a HeLa or 293 T HEK cell. . . '

The invention further provides said method wherein the promoter is a human CMV promoter.

The invention further provides a caspase-8 reactive peptide comprising at least 4 consecutive amino acids of a protein of the invention.

The invention also provides a derivative of said peptide. Furthermore, the invention provides said peptide derivative which is capable of forming a covalent bond upon contact with caspase-8.

The invention further provides a ribozyme specific for the nucleotide sequence corresponding to the DNA sequence of the invention.

The invention further provides a nonsense (antisense) oligonucleotide comprising at least 9 nucleotides of a sequence corresponding to the DNA sequence of the invention.

The invention further provides antibodies directed to an epitope of a protein of the invention.

The invention further provides an immunoassay suitable for detecting a caspase-8 interacting protein using the antibodies of the invention.

The invention further provides an immunoassay suitable for detecting a caspase-8 interacting protein using a peptide of the invention.

The invention further provides an immunoassay useful for detecting caspase-8 using a protein of the invention.

The invention further provides a method for identifying caspase-8 interacting proteins comprising:

(a) a yeast cell containing a reporter gene linked to a promoter containing a DNA sequence motif;

b) expressing the p20 subunit of said ksapase-8 in said yeast cell,

c) expressing a fusion protein of the DNA binding domain and the p10 and / or p20 subunit of said caspase-8 in a yeast cell, wherein said DNA binding domain is capable of binding to a motif of said DNA sequence,

d) expressing the non-fused p10 or p20 subunit of said caspase-8 in said yeast cell,

e) transforming the culture of said yeast cell with a library comprising an expression vector directing the expression of the fusion protein, the vector comprising a cDNA library and a transcriptional activator,

f) testing the culture of transformed yeast cells, selecting cells where the reporter gene is activated,

g) isolating the yeast cells of step f) and further isolating the caspase-8 interacting protein expressed in its vector.

The invention also provides a caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative thereof, said ribozyme, nonsense oligonucleotide, or antibody, which are suitable for use in modulating caspase-8 activity.

The invention further provides said caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative thereof, said ribozyme, nonsense oligonucleotide or antibody, which are useful for use in Fas-mediated modulation.

TNF-receptor or

The invention further provides a protein interacting with said caspase-8, an isoform, allele variant, fragment, functional analog, mutant or derivative thereof, said ribozyme, a nonsense oligonucleotide or antibody suitable for use in modulating apoptosis.

The invention provides a caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative thereof, said ribozyme, a nonsense oligonucleotide, or an antibody, which are suitable for use as a medicament.

The invention further provides a caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative thereof, said ribozyme, nonsense oligonucleotide or antibody, which are suitable for use as a medicament in the treatment of multiple sclerosis with primary oligodendrogliopathy, autoimmune uveoretinitis psoriasis, autoimmune myocarditis I, chronic mediated HCV, chronic castritis such as gastritis type A, mixed connective tissue disease (MCTD), Crohn's disease or ulcerative colitis.

diabetes, hepatitis

The invention further provides a triplex-forming oligonucleotide which is capable of binding to a sequence in the promoter region of a purine-rich caspase-8 interacting gene. The triplex-forming oligonucleotide may contain chemical treatments such as N, N-diethylethylenediamine-treated internucleoside phosphate bonds, uridine, benzo [g] -quinazoline-2,4-dione (1H, 3H) -dione or benzo [f] - quinazoline-2,4-dione- (1H, 3H) dione residue in place of thymidine residues. The triplex-forming oligonucleotide may further covalently bind to chemical agents that exhibit affinity for DNA. Preferred are agents that can be incorporated into DNA, such as acridine and psoralen.

The term "interacting" related to the interaction of a protein of the invention with caspase-8 means direct forms of interaction such as binding, cleavage and indirect interaction, for example, through adapter proteins. The interaction may lead to modulation of the caspase-8 mediated signal.

The term "binding related to the binding of caspase-8 interacting proteins" refers to the physical association of the caspase-8 interacting protein. Physical association can be measured by immunoassays such as ELISA or RIA, in a two-hybrid assay, immunoprecipitation assays or in assays based on size separation, such as acrylamide gel electrophoresis under non-denaturing conditions, or size exclusion gel chromatography. caspases or subunits thereof and protein of the invention. When using a two-hybrid assay, it is clear that caspase-8 or its subunit is expressed as a fusion of the DNA activation domain and the caspase-8 interacting protein is expressed as a fusion of the DNA binding domain or vice versa.

The term "two-hybrid assay" refers to a two-hybrid assay wherein protein-protein interactions can be measured by introducing a first expression vector that encodes the fusion of the first protein and the DNA binding region into yeast cells and a second expression vector that encodes the fusion of the second protein with the DNA activation domain. The yeast cells must contain at least one promoter-driven reporter gene containing a DNA sequence motif which is recognized by said DNA binding region. Usually, two reporter genes are used, for example histidine synthetase and beta-galactosidase. This makes it possible to prevent false positive results that may arise from mutations. This method is modified to use, for example, in the WO document, the location of both fusion in the assay, protein isolation, TNF-R and Fas signals (see 97/03998. Two hybrid assays consist of proteins in the cell nucleus.

The term " two-hybrid assay further includes modification of the two-hybrid method using ras and sos cell growth signaling proteins (Broder et al., Curr Biol. 1998, 8, p. 1121-4, Aronheim et al., Nucleic Acids Res. 1997, 25 p. 33734). For this protein to be functional it requires a localization to the cell membrane. This is achieved by expressing a cell growth signaling protein, such as a fusion with the first protein, and expressing a cell membrane localization signal. Such as myristilization of the signal sequence and fusion with a second protein. The interaction between the first and second proteins will result in the localization of the cell growth signaling protein to the cell membrane, triggering cell growth. Aggressively growing cells were then selected for further study. This system differs from the two-hybrid system described above because it does not depend on the location of both hybrids in the cell nucleus.

The term "bait" means a protein in the above two hybrid assays, which is expressed as a fusion protein with a DNA binding region.

The term "predator" refers to a protein in the two-hybrid assay described above, which is expressed as a fusion to the DNA activating region.

Many molecular biology methods are not described in detail here, as they are generally known in the art. Such methods include site-directed mutagenesis, PCR cloning, phage library screening using oligonucleotide or cDNA probes, recombinant protein analysis, bacterial and yeast cell transformation, mammalian cell transformation, and the like. These methods are described in Sambrook et al., Molecular •

Cold Spring Harbor Laboratory Cloning A Laboratory Manual,

ISBN: 0879693096, Current Protocols in Molecular Biology

M. Ausubel, ISBN 047150338X, 1988 and Short Protocols in

Molecular Biology, FM Ausubel et al. (Eds.) ^3rd ed. John

Wiley and Sons, ISBN: 0471137812, 1995.

A two-hybrid or three-hybrid system was used to identify caspase-8 interacting proteins and their potential substrates by the two-hybrid assay method.

The method of the invention uses a two-hybrid system essentially as described in Fields and Song, Nature 340, p. 245 (1989). Individual vectors, yeast strains and libraries can be obtained from Clontech (Palo Alto, USA) as components of the two-hybrid Matchmarker system (# PT1265-1).

A preferred embodiment of a two-hybrid yeast system as used in the methods of the invention is described in Boldin et al., Cell. 85 p. 803-15, 1996. The yeast two-hybrid system is further described in US Patent 5,580,736 (Brent et al.).

The tri-hybrid system is essentially used as described by Tirode et al., J. Biol. Chem. 272, p. 22995-9, 1997. When detecting caspase-8 interacting proteins of the invention, it is necessary that the two caspase-8 subunits be expressed independently. For this reason, the two caspase-8 subunits were preferably expressed separately, with different promoters driving the expression. In a preferred embodiment of the invention, the p10 caspase-8 subunit (serine at position 375 to aspartic acid at position 479) was expressed under the control of a weak yeast functional promoter within the DNA binding region. The weak promoter is the yeast ADH promoter and the DNA binding protein is the yeast Gal-4 transcriptional activator DNA binding region or the bacterial LexA protein.

The ADH promoter and the Gal4 DNA binding region are present, for example, in pGBD, which is commercially available from Clontech, Palo Alto, USA. However, it will be appreciated that other promoters may be used as long as they are active in yeast cells. Other DNA binding regions may also be used as long as these DNA binding regions do not activate transcription.

The long, active subunit of caspase-8 p20 (serine at position 217 to aspartic acid at position 374) is expressed as an unfused protein, under the control of an inducible promoter that functions in yeast cells. The use of the methionine suppressible Met25 promoter is preferred, as described by Tirode F. et al., J. Biol. Chem. 1997, 272 (37): 22995-9. The use of an inducible promoter simplifies the detection of the p10-p20 complex by immunoprecipitation and polyacrylic gel electrophoresis. The inducible promoter has other advantages. In yeast, it allows the expression of a protein which can be toxic to yeast cells, since there is a possibility to limit the time at which a potentially toxic protein is expressed.

The proteins to be tested by the method of the invention are produced in the form of a cDNA library. However, genomic and combined libraries can also be used. The library is cloned in yeast to the C-terminus of the transcriptional activation region. It is preferred that the transcriptional activation region of the yeast Gal-4 protein be used, but a large number of other transcriptional activators may be used. Preferably, the pGAD GH vector, available from Clontech, is used for cloning the library.

The yeast strain used for testing must contain a selection marker, such as a heptidine synthetase. It further comprises a promoter which comprises a DNA sequence to which the above DNA binding region specifically binds. The yeast cell preferably also contains a reporter gene driven by the yeast cell. a promoter comprising a DNA sequence to which the aforementioned DNA binding region specifically binds. For testing Gal-4 binding region hybrids, the yeast strain HF7c available from Clontech may be used. The L40 strain can be used when the DNA binding region is lexA.

After transformation, the yeast cells are placed under conditions that are selective. They are plated on medium plates lacking certain amino acids that are necessary for the stability of plasmids introduced into cells.

The medium is selective for yeast cells in which the aforementioned selection marker is activated. It is preferred that the selection marker is a histidine synthetase gene. Yeast cells expressing this gene can be selected by culturing on a medium lacking histidine. An advantage of this system is that the histidine synthetase inhibitor 3-aminotriazole can be added to the growth medium. It is also possible to inhibit the growth of yeast cells in which a small amount of histidine synthetase is expressed, causing removal of a promoter containing the sequence to which the above-mentioned DNA binding region specifically binds. In some clones, nonspecific interaction between the p10 subunit of caspase-8 and / or p20 and said clone can cause false activation of said promoter. By increasing the concentration of the inhibitor in the medium used to select the interaction clones, only clones that interact with a certain minimum force can be selected. It is preferred that the concentration of 3-aminotriazole be 7.5 mM.

Clones identified by their ability to grow on media that do not contain histidine were further analyzed by quantifying their reporter gene activity. The lacZ gene is preferably used as a reporter gene. Quantification of lacZ activity to culture as described

Chem. 270, 7795-8 (1995).

is preferably carried out in liquid by Boldin et al., J. Biol

The test methods described above can be performed similarly using a two-hybrid assay. A significant difference is that the two caspase-8 subunits are expressed as a single peptide chain which is a fusion protein containing the DNA binding region described above. The p20 subunit is mutated at the active site (cysteine at position 360-serine at position 360) as described below.

The p10 subunit is preferably separated from the p20 subunit by a linker, preferably located between amino acids at positions 10 and 50. Preferably, the linkers comprise small uncharged amino acids such as glycine, serine, threonine, alanine and valine. The linker preferably comprises serine and glycine residues. Most preferably, the ratio between glycine and serine residues in said linker is about 3: 1 to 4: 1.

Obtaining clones using the two-hybrid caspase-8 bait described above is similar to the three-hybrid system except that clones that bind only to one of the subunits cannot be distinguished from clones that bind to both or require subunits to bind a complex of both subunits. Any clone that appears using a two-hybrid assay can easily be tested for binding to two subunits by assessing the activity of lacZ double transformants, i.e., yeast cells that have been transformed with the newly identified clone and the p10 or p20 subunit of caspase-8, wherein said subunit forms fusion with a DNA binding region. Binding to the p10 and p20 subunit complex can be readily assessed by determining the activity of the lacZ triple transformants, where one of the caspase-8 subunits is expressed as a fusion protein with a binding region of " fc " • · · ··· · fcfcr

DNA and other subunits are expressed as a non-fusion protein. Obviously, the above three-hybrid system can therefore be used to determine the binding characteristics of the clone found by the two-hybrid system. The inducible promoter used allows rapid identification of clones interacting only with the p10 subunit or with the p10-p20 subunit complex.

For further study, clones were selected that were able to grow in a histidine-free culture medium that express lacZ activity. First, the proteins encoded by the clones are tested for their ability to bind irrelevant porteins, such as lamin. Generally, lamin binding clones are removed.

Clones found to specifically interact with caspase-8 are then further analyzed. This is also done with partial clones that were obtained directly by the above-described assay methods, as well as with full-length clones that were obtained based on the sequence of said partial clones. In order to obtain full-length clones, the partial clone sequence was obtained by extracting the DNA of said clone from yeast cells by methods well known in the art. The DNA is then transformed into bacteria to yield a large amount of purified DNA that can be used for sequencing. Alternatively, an insert into a vector, preferably the above-mentioned pGBD vector, can be excised using restriction enzymes and cloned into another vector for sequencing, such as pBluescript, available from Stratagene. Sequencing is performed by a chain termination method, preferably using the enzyme Sequenase 2. For example, a sequencing kit from United States Biochemicals may be used.

The sequences thus obtained can then be inserted into a database search program, and the overlapping sequences are identified by searching the database. Usage programs are well known in the art and include, for example, the GCG (genetics Computer group) package. A search engine such as the Basic Local Alignment Search Tool (BLAST), which is available from the EMBL server, is preferred (for example, http://dove.embl-heidelberg.de/Blast2). The Blastn command can be used to search for nucleotide sequences that overlap or are similar to the identified clone.

The protein identified by the method of the invention is prepared in the form of a fusion protein with a DNA binding domain. Therefore, the framework within which the nucleic acid sequence could be translated must be within the coding sequence of the DNA binding region. The DNA sequence of the clone identified by the invention can therefore be translated into an amino acid sequence. The Blast program, obtainable from the above-mentioned EMBL server, can then be used to identify overlapping sequences of a protein or similar proteins.

Alternatively, or in addition to the above library search methods, a library or library, such as a genomic cDNA, may be screened to identify the entire clone. Such assay methods are described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN 0879693096, Current Protocols in Molecular Biology, FM Ausubel, 1988, and Short Protocols in Molecular Biology, 1988; FM Ausubel et al. (eds.) ^3rd ed. John Wiley and Sons, ISBN: 0471137812, 1995. Alternatively or additionally, PCR-based cloning methods such as rapid amplification of cDNA ends (5 'and 3'RACE, Graham et al., Biochem. Biophys. Res. Commun. 177: 8-16 (1991).

Partial clones were identified by the assay of the invention, or full-length clones were obtained by any of the above methods. These clones were further screened. This is done, for example, by testing the sensitivity of these clones to proteolytic cleavage by active caspase-8. This assay can be performed in vivo. The mammalian cell line is then transfected with an expression vector that produces a protein encoded by the test clone and an expression vector encoding a second protein whose expression will induce caspase-8 activity. Expression vectors preferably contain a strong promoter suitable for expression of a clone and a second protein, such as the promoter of Rous sarcoma virus (RSV, Yamamoto et al., Cell 22, p. 787-97, 1980), myeloproliferative sarcoma virus (MPSV, Artelt P. et al., Gene 68, p. 213-9, 1988), cytomegalovirus (CMV, Thomsen, et al., PNAS. 82 p. 659-63, 1984) or similar promoters of viral and cellular origin.

The second protein whose expression identifies caspase-8 activity was selected from the group consisting of the intracellular domain of Fas, the intracellular domain of CD120a, Mort-1, caspase-8, or the equivalent of a protein capable of inducing caspase-8 activity. Alternatively, caspase-8 activity can be induced in cells by treatment with TNF or CD95 ligand. Experiments that examine the details of a possible mechanism and other technical details of this type of assay are described in Boldin et al., Cell 1996, supra.

Upon introduction of the expression vector coding for the second protein, the test protein is introduced into a mammalian cell and the cell culture is cultured for a period of time sufficient to allow protein expression, activation or expression of caspase-8 and cleavage. test protein. Said time is usually 4 to 72 hours, preferably 16 to 30 hours, most preferably 20 to 24 hours. Whole cell lysate is prepared to determine the extent of cleavage. Alternatively, the labeled protein can be purified using anti-label antibodies or nickel nitrilotriacetic acid chromatography, and further using reagents and procedures available from Qiagen GmbH, Hilden, Germany. The method of immunoprecipitation is described in Boldin et al., Cell, 1996, supra.

Immunoprecipitation reagents and instructions available from Boehringer Mannheim, Mannheim, as a kit.

are Germany in Germany

Whole cell lysate of purified protein is now resolved by size on an SDS polyacrylic gel by electrophoresis.

The test protein or labeled fragments thereof can now be visualized by Western blotting using anti-label antibodies. A preferred label is the histidine tail in combination with anti-histidine antibodies. However, other combinations of a labeling sequence and specific antibodies may be used as long as the antibodies remain specific for the labeling sequence. That is, they do not recognize other proteins in whole cell lysates. The specificity of the cleavage can be verified by control reactions where a specific caspase inhibitor is added to the mammalian cell culture for the above-mentioned time period. A preferred inhibitor is an inhibitor selected from the group consisting of zVAD-fmk, zDEVD-fmk, zIETD-fmk (Keppler-Hafkemeyer et al., Biochemistry 37, p. 16934-42, 1998). A more preferred inhibitor is zVAD-fmk. Other inhibitors that can then be used are proteins such as Bclx or p35, which acts as a cellular caspase inhibitor. Inhibition of cleavage where such proteins are co-expressed in the assay indicates that the cleavage is caspase-specific.

A second assay suitable for determining whether a test protein is cleaved by caspase-8 is an in vitro assay using recombinantly produced caspase-8 in an enzymatic reaction together with a labeled protein. The test protein can be produced in the manner described hereinabove. ≪ tb > ______________________________________ < tb > ______________________________________ < tb > by cloning the coding sequence into an expression vector that contains a strong promoter and transfected into a mammalian cell. It is preferred that the test protein is labeled as described above and can thus be isolated from a mammalian cell extract by means of anti-label antibodies or by using other reagents that are capable of specifically binding to a label sequence. Alternatively, the test protein can be produced in vitro using an in vitro translation system. The in vitro translation technique is well known in the art and reagents and detailed protocols are available from Stratagene, La Jolla, USA.

Alternatively, the test protein may be labeled using, for example, a radioisotope. Preferably, it is used for isotope labeling that the test protein is expressed in vitro and an isotope-labeled amino acid is added along with the unlabeled amino acid during the in vitro translation reaction. The isotope S ³⁵ is preferred. It is further preferred that the labeled amino acid was ³⁵ S methionine and the ratio between labeled and unlabeled amino acid was 1: 1 to about 1: 1 000th

The recombinantly produced test protein and the recombinantly produced active caspase-8 enzyme are then combined in a suitable buffer and for a time sufficient to allow cleavage. Preferred buffer and other preferred assay parameters are described in Boldin et al., Cell 1996. The preferred time is preferably between 10 minutes and several hours. Preferably, 30 minutes to one hour is preferred.

After cleavage has occurred, the reaction mixture is resolved by size distribution on an SDS polyacrylic gel by electrophoresis. If isotope labeling is used, the gel is dried and the isotope is detected by exposure to a photographic film or phosphoimage (Fuji). The test protein is labeled and may be

detected by Western blotting.

labeling specific antibodies

The discovery of another low molecular weight band in the reaction mixture to which the ksapase-8 protein was added compared to the control reaction mixtures not containing caspase-8 indicates cleavage of the test protein by caspase-8. In addition, the size of the low molecular weight band indicates the approximate position of the cleavage site.

The invention further provides a DNA sequence encoding caspase-8 interacting proteins.

The invention further encompasses DNA sequences encoding a biologically active isoform, allele variant, fragment, functional analog, mutant or derivative of caspase-8 interacting protein and a protein, isoform, allele variant, fragment, functional analog, mutant or derivative encoded thereby. Such analogs, fragments, mutants and derivatives are prepared in a standard manner (see, e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology, 1989), in which: For example, a DNA sequence encoding a caspase-8 interacting protein, one or more codons may be deleted, added, or substituted with another sequence to produce analogs that comprise at least one altered amino acid residue with respect to the native protein.

The invention also provides the above DNA sequences which encode a caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative, further comprising DNA sequences capable of hybridizing to a cDNA sequence derived from the coding region of a natural caspase interacting protein. -8, wherein said hybridization proceeds under moderately stringent conditions and the hybridizable sequence encodes a biologically active protein interacting with caspase-8. These hybridizable DNA sequences therefore include DNA sequences that exhibit relatively high homology to the natural cDNA sequence of the caspase-8 interacting protein and as such represent caspase-8 interacting protein-like sequences, which may, for example, be naturally derived sequences encoding different isoforms of the interacting protein caspase-8 or naturally obtained sequences encoding various isoforms of caspase-8 interacting protein or naturally occurring protein coding sequences belonging to a family of caspase-8 interacting protein sequences encoding a protein having caspase-8 interacting protein activity. . Further, the sequences may also, for example, include non-naturally occurring, synthetically produced sequences that are similar to the natural caspase-8 interacting cDNA sequence but incorporate a number of modifications desired. Such synthetic sequences therefore include all possible sequences encoding analogs, fragments, and derivatives of caspase-8 interacting protein, all of which have caspase-8 interacting protein activity.

The stringent hybridization conditions are determined by the temperature of the hybridization experiment, the molarity of the monovalent cations and the percentage of formamide in the hybridization solution. To determine the degree of stringency of the conditions, the equation of Meinkoth et al. 1984, determining the stability of hybrids showing 100% identity and expressed as the half-denaturation temperature of the T _ra DNA-DNA hybrid:

T _m = 81.5 ° C + 16.6 (logM) + 0.41 (% GC) -0.61 (% form.) - 500 / L, where M is the molarity of monovalent cations,% GC is the percentage of nucleotides G and C in DNA,% form. is the percentage of formamide in the hybridization solution and L is the length of the hybrid expressed in pairs of bases. For each 1 ° C by which the T _m calculated for a hybrid showing 100% identity is reduced, the amount of mismatch is increased by one percent.

Thus, if the T _m used in any hybridization experiment at a specified salt and formamide concentration is 10 ° C lower than the T _m calculated for a 100% Meinkoth hybrid, hybridization occurs even if there is up to about 10% mismatch .

“Medium stringency conditions that provide a T _m that is no more than 20 ° C below the T _m that would exist for a perfect duplex with the target sequence. This temperature was calculated either according to the above formula or was actually measured. Under moderately stringent conditions (15-20 ° C below calculated or measured T _m hybrid), a 2 x SSC (standard physiological saline citrate) wash buffer and 0.5% SDS (sodium dodecyl sulfate) at a suitable temperature below the calculated wash buffer is used. T _m for hybrid. The limiting stringency of the conditions is primarily due to the wash conditions, especially if the hybridization conditions used are those which, together with stable hybrids, allow the formation of less stable hybrids. Washing conditions at higher stringency then remove less stable hybrids. Conventional hybridization conditions that can be used under moderate stringent wash conditions that include solution hybridization (standard physiological solution described above, 6x SSC (or 6x SSPE solution-phosphate-EDTA)), 5x

Denhardt's reagent, 0.5

SDS, 100 .mu.g / ml of denaturant, cleaved salmon sperm DNA at a temperature that is approximately 20-25 ° C below T _m. If a mixture of probes is used, it is preferred to use tetramethylammonium chloride (TMAC) instead of ₍ SSC (FM Ausubel, ISBN: 047150338X, 1988 and Short Protocols in Molecular Biology, FM Ausubel et al., Eds.) 3 ^rd ed. John Wiley and Sons, ISBN: 0471137812, 1995).

In order to obtain the various naturally occurring caspase-8 interacting protein sequences listed above, standard procedures for testing and isolating naturally obtained DNA and RNA samples from various tissues can be performed. using naturally occurring caspase-8 interacting cDNA or a portion thereof as a probe (see, e.g., Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current). Protocols in Molecular Biology (1989).

The invention provides a caspase-8 interacting protein, which may be defined by the assay described above. The invention also provides a polypeptide or protein substantially corresponding to a caspase-8 interacting protein. The term " substantially corresponding " includes not only caspase-8 interacting protein but also polypeptides or proteins that are analogues thereof.

Analogues substantially corresponding to caspase-8 interacting protein are those polypeptides in which one or more amino acid sequences of the caspase-8 interacting protein have been replaced by another amino acid, deleted and / or incorporated, the resulting protein having substantially the same or higher biological activity than the caspase-8 interacting protein to which it corresponds.

In order for a protein to correspond to a caspase-8 interacting protein, changes in the sequence of caspase-8 interacting proteins, such as isoforms, are generally relatively small. Although the number of changes may be greater than 10, no more than five are preferred, and no more than three such changes are most preferred. While it can be used to find potentially biologically active proteins interacting with caspase-8 by any method, one such method is to use a conventional DNA mutagenesis method that encodes a protein, resulting in only a few modifications. Proteins expressed by such clones can then be tested for their ability to bind to caspase-8 and to modulate caspase-8 activity by modulating the intracellular pathways described above.

“Conservative changes are those that are not expected to alter protein activity and are usually tested first and as such do not substantially alter the size, charge and configuration of the protein and will not alter the biological properties of the protein. Conservative substitutions of caspase-8 interacting proteins include an analogue wherein at least one amino acid residue in the polypeptide is conservatively replaced by a different amino acid. Such substitutions are preferably made in accordance with the following list in Table IA. These substitutions can be determined by routine experimentation to produce modified structural and functional properties of the synthesized polypeptide molecule while retaining the biological activity of the caspase-8 interacting protein.

Table IA:

original remainder examples of substitutions

Ala Gly; Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gin Asn Glu Asp Gly Ala; Pro His Asn; Gln Ile Leu Val Leu Ile Val Lys Arg; Gln; Glu Met Leu; Tyr; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Php Wall Ile, Leu

Another class of caspase-8 interacting protein substitutions are those in which at least one amino acid residue in the polypeptide has been deleted and a different residue has been inserted in its place in accordance with Table IB. The types of substitutions that can be made in a polypeptide can be based on the analysis of amino acid change rates between homologous proteins of different species, such as those presented in Tables 1 and 2 of Schulz et al., GE Principles of Protein Structure Springer- Verlag, New York, NY, 1798 and in Figures 3 to 9 of Creighton, T. E., Proteins: Structure and Molecular Properties, WH Freeman and Co., San Francisco, CA 1983. Based on such analysis, alternative conservative substitutions are defined as changes in one of the following five groups:

Table No. 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, Gin,

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

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

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

The three amino acid residues in parentheses have special roles in protein construction. Gly is the only residue that contains no side chain and thus guarantees flexibility for the chain. This fact favors the creation of a 'secondary structure that differs from the alpha-helical structure. Because of its unusual geometry, it connects the chain and generally tends to support beta-like loop structures, although in some cases Cys may be able to participate in

• Disulfide bridge formation, which is important for protein folding. It should be noted that in Schulz et al., Groups 1 and 2 are combined. It should be further noted that Tyr, due to its hydrogen-binding potential, is essentially related to Ser and Thr.

Conservative amino acid substitutions of the invention, for example, those described above, are well known in the art and are expected to retain biological and structural properties upon amino acid substitution. Most of the deletions and substitutions of the invention are those that do not produce radical changes in the characteristics of the protein or polypeptide molecule. The term "characteristics" is defined in a non-exclusive manner to define both changes in the secondary structure, such as alpha-helix or beta-leaf, as well as changes in biological activity, such as binding to caspase-8 and / or mediating the effect of caspase-8 on cell death .

Examples of producing amino acid substitutions in proteins that can be used to obtain analogs of caspase-8 interacting proteins when used in the present invention include any of the methods set forth in US Patent Documents. RE 33,653, 4,959,314, 4,588,585 and 4,737,462 (Mark et al.), 5,116,943 Koths et al., 4,965,195 Namen et al., 4, 879,111 Chong et al., And 5,017,691 Lee et al., And lysine-substituted proteins that described in U.S. Patent No. 4,904,584 (Shaw et al.).

In addition to the conservative substitutions discussed above and which do not substantially alter the activity of the caspase-8 interacting protein, the invention describes conservative substitutions or less conservative substitutions and more random substitutions that result in increased biological activity of caspase-8 interacting protein analogs.

· · ·

When determining the exact effect of substitution or deletion, it is desirable that the effect of substitution, deletion, etc. be evaluated by routine binding and cell death assays.

Acceptable non-interacting caspase-8 analogues are those that retain at least the ability to interact with caspase-8, thereby mediating caspase-8 activity in the intracellular pathway or modulating caspase-8 alone activity. In this way, analogs having a so-called dominant negative effect can be produced, namely an analog that either does not bind to caspase-8 or is deficient in subsequent signaling or other activity following binding. Such analogs may be used, for example, to inhibit the cytotoxic effect of caspase-8 or to increase cell death or cell survival depending on which of these activities is the major modulated interaction of caspase-8 interacting protein (described above) and such analogs then compete with the natural protein interacting with caspase-8 for binding or interaction with caspase-8.

At the genetic level, these analogs are genetically prepared by site-directed mutagenesis of nucleotides into DNA that encodes a caspase-8-interacting protein, producing DNA encoding the analog, then synthesizing the DNA and expressing the polypeptide in recombinant cell culture. Analogs typically exhibit the same or increased qualitative biological activity as a naturally occurring protein (Ausubel et al., Current Protocols in Molecular Biology, Greene & Wiley Interscience, New York, NY, 1987-1995, Sambrook et. al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

Accordingly, the preparation of caspase-8 interacting protein or alternative nucleotide sequences encoding the same polypeptide but different from the native sequence, due to changes that are made possible by the degeneracy of the genetic code, can be accomplished by site-directed mutagenesis of the DNA that encodes previously a prepared analogue or natural version of a caspase-8 interacting protein. Site-specific mutagenesis allows the production of analogs by using specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of contiguous nucleotides to generate a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion. Typically, a primer of about 20 to 25 nucleotides is preferred, with about 5 to 10 complementing nucleotides being altered on each side of the sequence. In general, the site-specific mutagenesis technique is well known in the art and is described in Adelman et al., DNA 2: 1983.

The site-directed mutagenesis method typically uses a phage vector that exists in both single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as M13 phage, such as described in Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant DNA, edited by A. Walton, Elsevier, Amsterdam (1981). These phages are commercially available and their use is well known in the art. Alternatively, plasmid vectors that contain a single stranded phage origin of replication can be used to obtain single-stranded DNA (Veira et al., Math. Enzymol. 153: 3, 1987).

Site-directed mutagenesis in accordance with the invention was accomplished by first obtaining a single-stranded vector that includes in its sequence a DNA sequence that encodes a relevant polypeptide.

• 4 * 4 * 4 4 4 4 4 4 4 4 4 4

Oligonucleotide. a primer carrying the desired mutated sequence was prepared by synthetically automated DNA / oligonucleotide synthesis. This primer was then coupled to a vector containing a single chain protein sequence and polymerization enzymes such as the Klenow fragment of E. coli polymerase I were used to synthesize the mutant-carrying chain. Thus, the mutated sequence and the second strand carry the desired mutation. This heteroduplex vector was then used to transform suitable cells, such as E. coli JM101 cells, and clones were selected that include recombinant vectors carrying the mutated sequence alignment.

After such a clone has been selected, the mutated sequence of the caspase-8 interacting protein can be removed or replaced with a suitable vector. Generally, it is a type of transfer or expression vector that can be used to transfect a suitable host.

The gene or nucleic acid encoding a caspase-8 interacting protein can also be detected, obtained and / or engineered in vitro, in situ and / or in vivo using a known method of DNA or RNA amplification, such as PCR and chemical oligonucleotide synthesis. PCR allows the amplification (increase in number) of specific DNA sequences by repeated DNA polymerase reactions. This reaction can be used as a replacement for cloning. All that is required is knowledge of the nucleic acid sequence. In order to perform PCR, it is necessary to design primers that are complementary to the sequence. 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 designed such that complementary mismatches can be tolerated. Amplification of these regions of mismatch can lead to the synthesis of a mutagenized product resulting in a peptide with new properties (i.e., site directed mutagenesis) * «• · (described in the publication, FM Ausubel et al. (Eds.) ^3rd ed. John Wiley and Sons, ISBN: 0471137812, 1995, Ch. 16).

Coupling synthesis of complementary reverse transcriptase with PCR is the starting material for synthesis

DNA (cDNA) using RNA can be used as extracellular regions of the prolactin receptor without the need for cloning.

PCR primers can be designed to contain new restriction sites and other features, 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 caspase-8 interacting protein or fragment thereof to be designed to be suitable for ligation of other sequences and / or cloning sites in the vectors.

PCR and other methods of RNA and / or DNA amplification are well known in the art and can be used according to the invention without having to carry out experiments with the knowledge and instructions provided herein. Known methods for amplifying DNA or RNA include, but are not limited to, polymerase chain reaction (PCR) and related amplification procedures (e.g., U.S. Patent Nos. 4,683,195, 4,683,202, 4,800,159,

Nos. 4,965,188 (Mullis et al.), 4,795,699 and 4,921,794 (Tábor et al.

5,122,464 et al., 4,889,818 4,766,067 al., 5,142,033 (Innis), 5,122,464 (Wilson)

5,091,310 (Innis), 5,066,584 (Gyllensten et al.), (Gelfand et al.), 4,994,370 (Silver et al.), (Biswas), 4,656,134 (Ringold) and Innis et al., Eds., PCR Protocols: RNA-mediated amplification that uses antisense RNA with the target sequence as a template for double-stranded DNA synthesis (US Patent No. 5,130,238 (Malek et al., NASBA) and immuno-PCR, which combines the use of amplification DNA labeled with antibodies (Ruzicka et al.)

Science 260: 487 (1993), Sano et al., Scince 258: 120 (1992),

Sano et al., Biotechniques 9: 1378 (1991)).

Analogously, it is possible to prepare biologically active fragments of caspase-1 interacting proteins (for example fragments of caspase-8 interacting proteins or their interacting interacting isoforms) with caspase-1 with respect to

!. Suitable for protein analogs are caspase-8 protein fragments that retain the ability of a caspase-8 interacting protein and which may mediate the biological activity of caspase-8 or other caspase-8-associated proteins directly or indirectly. Fragments of caspase-8 interacting proteins can be prepared having a dominant negative or a dominant positive effect as described above with respect to said analogs. It should be noted that these fragments represent a special class of analogs of the invention, namely, portions of caspase-8 interacting proteins derived from the entire caspase-8 interacting protein sequence (for example, from the sequence of any caspase-8 interacting protein or isoform thereof). Each such portion or fragment has any of the above desired activity. Such a fragment may be, for example, a peptide.

Similarly, derivatives may be prepared by standard side group modifications of one or more amino acid residues of caspase-8 interacting protein, analogs or fragments thereof, or by conjugating caspase-8 interacting protein, analogs or fragments to another molecule such as antibody, enzyme, receptor, etc., which are well known in the art. The term "derivatives" means derivatives that can be prepared from functional groups that appear as side chains on residues or an N-terminal or C-terminal group in a manner well known in the art and are within the scope of the invention. The derivatives may have chemical moieties, such as carbohydrate or phosphate residues, to form a fraction showing the same or higher biological activity as the caspase-8 interacting proteins.

Derivatives may include aliphatic esters of carboxyl groups, carboxylic amides by reaction with ammonia or with primary or secondary amines, N-acyl derivatives or free amino groups of amino acid residues formed with acyl moieties (for example alkanoyl or carbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl groups (for example, a derivative of seryl and threonyl residues) formed by acyl moieties.

The term "derivatives" includes only those derivatives that do not change one amino acid for another of the twenty naturally occurring natural amino acids.

As described above, cleavage assays can be used to determine whether a caspase-8 interacting protein is cleaved by caspase-8. Separation based on the size of the digested fragments gives an approximate indication of the location of the cleavage site.

The cleavage sites can be further determined by preparing deletion mutants of the test protein, and each deletion mutant is tested for its susceptibility to caspase-8 cleavage as described above. Deletion mutants can be constructed by cloning PCR of the desired fragments of the test protein using the DNA sequence of the clone encoding said test protein as a template. Fragments amplified by PCR can then be cloned into expression vectors, where the ATG start codon and preferably the Kozak sequence must also be introduced (Kozak, M., Nucleic Acids Res. 12, p. 857-72, 1984). Further details of the expressing proteins can be found in the above-mentioned Qiagen information regarding His-tagged proteins, but also generally expressed protein. Protein expression is further described in Sambrook et al., Molecular Cloning & Laboratory Manual, Cold Spring Harbor Laboratory, ISBN 0879693096, Current Protocols in Molecular Biology, especially Chapter 16.

The cleavage site of the test protein can then be defined by preparing various deletion mutants and determining the smallest such deletion mutant that is cleaved by caspase-8.

peptide synthesis, Springer,

Another method for identifying cleavage sites uses peptides that are generated according to the predicted protein sequence of the test clone. Peptides can be synthesized chemically, for example, as described in Bodanzszky and Bodanszky, The Practice of Peptide Synthesis, Springer, New York, ISBN 0-387-13471-9 and Bodanszky, The Principles of New York, ISBN 0-387-12359 -4.

Peptide synthesis can be ordered from several commercial companies, such as SynPep Corp., Dublin, CA USA and California Peptide Research, Inc., Napa, CA, USA. The peptides can also be produced either as fusions with other proteins or unfused, by expressing recombinant DNA encoding them (Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096).

Current Protocols in Molecular Biology, especially in Chapter 16).

To use peptides to map the cleavage site of a test protein, the predicted amino acid sequence of said protein is divided into regions and a peptide corresponding to each region is synthesized. In addition, the peptides comprise about half the amino acids of one region, and about half of the amino acids that are directly adjacent to the synthesized region so that they overlap the boundaries between the two regions are joined to it. Surfaces contain between 5 and 100 amino acids, preferably between 9 and 40 amino acids, and most preferably «·

Λ Q * * · · · ··· ♦♦ ·

4Ζ · · «· _φ ···.

Prefers between 20 and 30 amino acids. A whole set of peptides is tested for susceptibility to caspase-8 cleavage as described above. The peptides prepared can be provided neat and in large quantities. The peptides can therefore be analyzed directly on SDS polyacrylic gel by electrophoresis and UV detection after the cleavage reaction, or visualized by staining, for example using Comassie blue. Alternatively, peptides can be labeled for ease of detection, for example, by an isotope (see, for example, Shevchenko A, et al., Rapid Commun Mass Spectrom. 11, 1015-24, 1997).

When peptide testing is completed, the peptide known to contain a caspase-8 recognition site can be further studied by repeating the same method, but selecting smaller regions selected from the sequence of the peptide that has been identified.

The current peptide cleavage site should confirm the caspase XXXD cleavage sequence (Boldin et al., Cel 1996 and Nicholson et al., Killer Caspases, Trends in Biochem Sci. 22, 299-306, 1997). The contribution of each amino acid in a peptide can be evaluated by preparing peptides that are mutated in one amino acid and testing these mutated peptides for susceptibility to caspase-8 cleavage. In order to mutate an amino acid, it is preferred to replace it with an amino acid selected from the group of charged non-polar amino acids (Lehninger, Biochemistry, Worth, NY, 1979, Chapter 4). Most preferably, the amino acid is selected from glycine or alanine.

By mutating critical amino acids, it is possible to generate peptides that bind to caspase-8 but are not susceptible to caspase-8 cleavage. Binding can be assayed by separation of peptide-caspase-8 complexes by size under conditions that are not denaturing using acrylamide gel electrophoresis.

• ·

Further, it is possible to construct engineered peptides that are capable of reacting with active cysteine of caspase-8, thereby covalently binding to said cysteine. Agents that react with thiol groups are well known in the art and can be used for this purpose. Such agents may bind in a reversible manner. For example, agents that contain a thiol group can react with the SH group of the active cysteine caspase-8. The formed S-S covalent bond can be cleaved by reduction, for example, under physiological conditions in the cytosol or by the use of an agent that reduces S-S groups, such as dithiothreitol (DTT). Agents that react with thiol groups may bind irreversibly to caspase-8. Such agents occur among crosslinkers capable of reacting with thiol groups as described in p. 0-90 and on other pages of the PIERCE Life Sciences catalog (PIERCE, Rockford, IL, USA).

Suitable groups which react with thiol groups are, for example, pyridyldithio, iodoacetamido or maleimido. These groups can bind to the peptide via linkers that contain unsaturated aliphatic hydrocarbon chains, -O-, -S-, -NH- or aromatic groups. Linkers may be substituted.

Thiol reactive groups can bind to the peptide by chemical synthesis as known in the art. Peptide functional groups can be reacted with suitable thiol-reactive linker molecule functional groups. A lysine residue present, for example, in a peptide sequence or added to form a suitable functional group which, in the case of lysine, is an epsilonamino group, can react with an agent such as an ester with a heterobifunctional N-gamma-maleimidobutyryloxysuccinimide crosslinking to form a peptide the crosslinking reacts with the N-hydroxysuccinimide reagents, while the maleimide group reacts with the N-hydroxysuccinimide reagent. The cross-linking agent remains unreacted and may, upon contact with caspase-8 cysteine, which occurs when said peptide is present in the cross-linking agent. specifically binds to said caspase-8, reacting with the thiol group of said cysteine, thereby deactivating said caspase-8.

The position in the peptide used in the reaction with the thiol-reactive agent may be selected to be close to the amino acid (usually aspartic acid) where the caspase-8 cleavage occurs. The linker by which the thiol-reactive group binds to the peptide may vary in length, by the presence of polar groups such as hydroxyl, charged groups such as nitro and sulfo, and aromatic groups such as phenylene phenyl residues. These differences in linker structure allow the formation of a reagent that contains a peptide and covalently bound to a thio-reactive agent that specifically binds to caspase-8 and effectively reacts with the thiol group of its active cysteine.

The test protein or peptide fragment thereof may be further characterized by introducing said protein or peptide into a mammalian cell and measuring the effect of apoptosis inducing agents in said cell.

Expression of the protein or peptide in a mammalian cell can be accomplished by incorporating DNA encoding the test protein into a vector that contains the promoter, intron sequence, and splice donor / acceptor signals and further comprises a termination sequence. These methods are generally described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology, Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology, Chapter 16.

• ·

The above promoter, intron, and termination sequences can be used in mammalian cells. The promoter is preferably a strong promoter such as the RSV, CMV or MPSV promoter mentioned above. The promoter may also be the early SV40 promoter (Everett et al., Nucleic Acids Res. 11 p. 2447-64, 1983) or a cellular promoter such as the beta-actin promoter or ELF-1 promoter (Tokushige). , et al., J. Virol. Methods, 64: 73-80 (1997). A hybrid promoter, such as a hybrid between the lac operator and the alpha promoter of human ELF-1, can also be used, as described in Edamatsu et al. , Gene 187, p. 289-94 (1997), the CMV beta-actin hybrid promoter (Akagi et al., Kidney Int. 51, p. 1265-9, 1997) or the hybrid between the tet operator sequences and the promoter CMV (Furth et al., PNAS 91, p. 9302-6, 1994).

Intron sequences that can be incorporated as complete sequences, i.e., include splice donor and acceptor sites, can be incorporated into the protein coding sequence that is required for expression. If such intron sequences can increase RNA stability, then they increase production of the desired protein. While in principle suitable intron sequences can be selected from any gene that contains introns, preferred intron sequences are the beta-actin intron, the SV 40 intron and the p55 TNF receptor intron.

The intron sequence may contain enhancer elements that can enhance transcription driven by the aforementioned promoters.

Intron sequences often contain transcriptional and translational control sequences that cause tissue-specific expression. If it is necessary to express the protein of the invention in a tissue-specific manner, it is preferred to use such intron sequences. An example of tissue-specific enhancer elements containing an intron is an erythroid-specific enhancer located in intron 8 of the human 5-aminolevulinate synthesis 2 gene (Surinya et al., J. Biol. Chem. 273, p. 16798-809, 1998). A discussion of the principle of enhancing protein production using intron sequences together with intron sequences is given in Huang et al., Nucleic Acids Res. 18, p. 937-47 (1990).

Transcriptional termination sequences and polyadenylation signals may be added to the 3 'end of the DNA encoding the protein to be expressed. Such sequences can be found in many and even most genes. It is preferred to use the SV 40 polyadenylation signal (Scheck et al., Mol. Cell Biol., P.5386-93, 1992).

A preferred vector suitable for expression of a protein in a mammalian cell is the pcDNAHis vector (Invitrogen), which contains a CMV promoter directing expression of the gene encoding the protein of interest. Other useful vectors include pCDNA3 or pMPSVEH vectors. These vectors contain CMV and MPSV promoters.

Using recombinant expression of the test protein, said protein can now be evaluated for its effect on caspase-8 mediated apoptotic signal. Apoptosis can be induced either by overexpression of an apoptosis inducing protein such as the intracellular domain of CD120a, the intracellular domain of CD95, the Mort-1 protein, caspase-8 or its equivalent, or activation of the apoptotic signal by triggering CD120a, CD95, TRAMP / DR3 or the equivalent receptor. Receptor activation can be achieved either by contacting the ligand receptors or by crosslinking the receptors with antibodies, preferably polyclonal antibodies (Engelmann et al., J. Biol. Chem. 265, p. 14497-504, 1990).

While generally triggering CD120a requires the addition of a protein synthesis inhibitor such as cycloheximide for apoptosis, overexpression to achieve a strong signal for receptor intracellular domains or proteins involved in the apoptosis signal is not required (Boldin et al., Cell 85, 803 (1996). The detection of apoptosis, incubation time and other details and parameters of this assay are described above by Boldin et al.

The death of cells that express the test protein, versus those that do not, can be evaluated by any method, such as methods based on DNA fragmentation or detection for apoptosis of specific antigens and epitopes. Reagents and protocols suitable for the detection of kit apoptosis are available from the aforementioned companies, for example, Boehringer Mannheim.

Cell death can also be determined by assessing the morphological appearance of the cells. Apoptotic cell death is also characterized by a corrugated cell membrane and cell shrinkage without lysis of the cells.

It is preferred that the expression gene be expressed in a mammalian cell in order to provide a marker for successful transfection. As such, the transfection process results in cell death, which includes death of cells that have not been transfected. It is preferred to evaluate only cells that have been transfected. A preferred reporter gene for this purpose is the lacZ gene, which can be easily detected by incubating the transfected Xgal cells with a similar agent that indicates active beta-galactosidase. However, it is possible to use any other reporter gene, preferably a gene whose genes can be easily detected using a simple color reaction, the results of which can be evaluated using a microscope. Green fluorescent protein can be used for direct detection without the need for a color reaction. This reporter gene makes it inevitable to use a fluorescent microscope.

• *

When considering only cells that have been transfected, i.e., those that express the reporter gene, and by counting the percentage of cells demonstrating apoptosis morphology, the effect of a particular transfected clone and the protein expressed in apoptosis can be evaluated.

Mammalian cells are preferably HeLa cells or human embryonic kidney cells (HEK) 293-T. Transfection is preferably performed by the calcium phosphate method as described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology. Cell morphology is evaluated once every 150 hours after transfection, preferably 4 to 35 hours after transfection, and most preferably 20 hours after transfection.

Preparation of antibodies

Polyclonal antibodies can be made using rabbits, chickens, mice, rats, sheep or the like mammals. In the preparation of antibodies against a protein or peptide of the invention, a protein or peptide is produced as described above by recombinant DNA technology in mammalian cells. The protein can also be produced in bacterial or insect cells, as described in more detail in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology.

The protein or peptide is isolated from the cells in which it is produced. Protein purification methods are well known in the art and are described in more detail in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology in Chapter 16 and Current Protocols in Protein Science, Wiley and Sons Inc., Chapters 5 and 6. It is preferred that the protein can be produced as a fusion with a second protein, such as glutathione-S-transferase or a similarly tagged sequence, such as a histidine sequence. The use of fusion or labeled proteins z simplifies the purification procedure as described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology, Chapter 16, and instructions for expressing labeled protein. his a in a cleaning kit from Qiagen.

If the protein or peptide is expressed as a fusion protein, it is necessary to remove the fusion partner by cleavage prior to using the antibody-producing protein to prevent antibody formation against the fusion partner. Cleavage of fusion partners and isolation of the desired protein is described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology, Chapter 16, supra. agents suitable for the expression and purification of maltose binding protein fused to recombinant proteins.

When producing the peptide of the invention, it is necessary to remove the fusion partner, whereby the fusion protein can be stimulated by producing antibodies against the peptide. In general, this consideration will be relevant when antibodies are produced from peptides that contain less than 50 amino acids.

As described above, the peptide can be further synthesized by chemical methods well known in the art.

The preparation of polyclonal antibodies against proteins is described in Chapter 2 of Current Protocols in Immonology, Wiley & Generation of antibodies against peptides may lead to some protocol changes due to generally lower antigenicity of the peptides compared to proteins. The generation of polyclonal antibodies against peptides is described in Current Protocols in Immunology, Chapter 9, supra.

Monoclonal antibodies can be prepared from B cells taken from the spleen or lymph glands of immunized animals, particularly rats and mice, by fusion with immortalized B cells under conditions that promote hybrid cell growth. In the case of fusion of murine B cells, the Ag-8 cell line is preferred.

A method for generating monoclonal antibodies is described in a number of articles and textbooks, such as Chapter 2 of Current Protocols in Immunology. Chapter 9 then describes immunization with peptides or animals. The spleen or lymph node cells of these animals can be used in the same way as the spleen or lymph node cells of animals immunized with a protein to produce monoclonal antibodies as described in Chapter 2 of that publication.

Methods used to prepare monoclonal antibodies are further described in Kohler and Milstein, Nature 256, 495-497 and in USP 4,376,110.

Preparation of antibodies from the human antibody gene bank is accomplished by replacing their hypervariable regions with nearly random sequences, as described in USP 5,840,479. Such antibodies are preferred when it is difficult to immunize an animal with a given peptide or protein. Some structures are weakly immunogenic and may thus remain despite addition of adjuvant and binding to other proteins in fusion constructs. Antibodies are described in USP 5,840,479. It is further preferred, if necessary, to use an antibody with a structure similar to human antibodies, for example when antibodies that exhibit low immunogenicity in humans are required.

• · · · · · · · · ·

Once suitable antibodies have been identified, their properties may need to be altered. For example, with chimeric antibodies, higher production yields can be achieved. Chimeric antibodies are required wherein the constant regions are replaced with constant regions of human antibodies and the antibodies have to be of low immunogenicity in humans. The preparation of chimeric antibodies has been described in a number of publications such as Cabilly et al., PNAS 81, p. 3273, 1984, Morrison et al.

al., PNAS 81, 6851, 1984, Boulianne et al., Nature 312, P · 643, 1984, EP 125023, EP 171496, EP 173494 EP 184187, WO 86/01533, WO 87/02671 a Harlow and Lane, Antibodies: AND

Laboratory Manual, Cold Spring Harbor Laboratory, 1988.

Another type of antibodies are anti-idiotypic antibodies. Anti-idiotypic antibodies (anti-Id) are antibodies that recognize single determinants that are generally associated with an antigen-binding site of antibodies. Antibody Id can be prepared by immunizing an animal of the same species and genetic type (e.g., a mouse strain) as a source of mAb to which anti-Id has been prepared. The immunized animal will recognize and respond to idiotypic determinants of immunizing antibodies by producing antibodies against these idiotypic determinants (anti-Id antibodies) (see, for example, US Patent No. 4,699,880).

Anti-Id antibodies can also be used as an "immunogen" to induce an immune response in another animal that produces so-called anti-anti-Id antibodies. Anti-anti-Id may be epitope identical to the original mAbs that induce anti-Id. Thus, using antibodies against idiotypic determinants of mAbs, it is possible to identify other clones expressing antibodies of identical specificity.

Monoclonal antibodies raised against caspase-8 interacting protein, analogs, fragments or derivatives of the invention can be used to elicit anti-Id antibodies in suitable animals such as BALB / c mice. Spleen cells from such immunized mice are used to produce anti-Id hybridomas that secrete anti-Id monoclonal antibodies. Furthermore, anti-Id mABs can be coupled to a carrier, such as hemocyanin of gastric gastropods (KLH), and used to immunize other BALB / c mice. Serum from these mice will contain anti-anti-Id antibodies, which may contain binding properties of the original mAbs specific for the epitope of the above-described protein interacting with caspase-8 or analogs, fragments and derivatives thereof.

Anti-Id monoclonal antibodies have their own idiotypic epitopes or "idiotypes" structurally similar to the epitope of interest.

The term "antibody" also includes both intact molecules as well as fragments thereof, such as Fab and F (ab ') 2, which are capable of binding antigen. Fab and F (ab ') 2 fragments do not contain an Fc fragment of intact antibodies, disappear very rapidly from the bloodstream, and may bind less non-specifically to tissue as compared to intact antibody (Wahl et al., J. Nucl. Med. 24: 316- 325 (1983)).

Suitably, Fab and F (ab ') 2 and other antibody fragments useful in the invention can be used to detect and quantify caspase-8 interacting protein according to the methods described herein for intact antibody molecules. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (in the case of Fab fragments) or pepsin (in the production of F (ab ') 2 fragments).

An antibody is capable of binding a molecule when it is capable of specifically reacting with the molecule, wherein the molecule binds to the antibody. The term " epitope " refers to a portion of any molecule capable of being bound to antibodies that the antibodies also recognize. Epitopes or "antigenic determinants" usually contain chemically active surface groups of molecules such as amino acids or carbohydrate side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics.

The term "antigen" is a molecule or portion of a molecule capable of being bound by antibodies that are additionally capable of eliciting in an animal by producing antibodies capable of binding to an epitope of said antigen. The antigen may have one or more epitopes. The specific reaction set forth above means that the antigen will react in a highly selective manner with the corresponding antibodies and not with a number of other antibodies that can elicit other antigens.

Antibodies comprising antibody fragments useful in the invention can be used to quantitatively or qualitatively detect caspase-8 interacting protein in a sample or to detect the presence of cells that express caspase-8 interacting protein of the invention. This can be done by immunofluorescence methods using fluorescently labeled antibodies (described below; associated with light microscopy, flow cytometry, or fluorometric detection).

Antibodies (or fragments thereof) useful in the invention may be used histologically, such as in immunofluorescence or immunoelectron microscopy, for in situ detection of caspase-8 interacting protein of the invention. Detection of the sine is performed by obtaining a histological sample from the patient, and such sample comes into contact with the labeled antibody. Preferably, the antibodies (or antibody fragment) are produced by applying a labeled antibody to or overlapping the sample with the antibody (or fragment thereof). Using such a procedure, it is possible to determine not only the presence of caspase-8 interacting protein, but also its distribution in the test tissue. Using the invention in the art, it will be appreciated that any of a variety of different histological methods (such as staining procedures) can be modified to achieve in situ detection.

Such assays suitable for caspase-8 interacting protein of the invention typically include incubation of a biological sample such as biological fluid, tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells that can be incubated in tissue culture in the presence of detectably labeled antibodies capable of identifying caspase-8 interacting protein and detecting antibodies by any number of methods well known in the art.

The biological sample may be applied to a solid support or carrier such as nitrocellulose or other solid supports or carriers that are capable of immobilizing cells, cell particles or soluble proteins. Such a support or carrier may then be washed with a suitable buffer followed by treatment with the detectable labeled antibodies of the invention as described above. The solid support or carrier is then washed a second time with the buffer to remove unbound antibodies. The amount of bound label on said solid support or carrier can then be detected by conventional methods.

The term "solid support," carrier, "solid support means any support or support capable of binding antigen or antibodies. A well known carrier or support includes, for example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbro and magnetite. The nature of the carrier may be either soluble or insoluble to some extent, according to the purpose of the invention. The support material may exhibit any possible structural configuration as long as the bound molecule is capable of binding to an antigen or antibody. The configuration of the substrate or carrier may be spatial, such as spheres, cylindrical, for example, the interior of the tube or the outer surface of the rod. Alternatively, the surface may be flat, such as a sheet or test strip, etc. Preferred substrates or carriers include polystyrene beads. Other carriers suitable for binding antibodies or antigens are well known in the art.

The binding activity of a given amount of antibodies of the invention can be determined by methods well known in the art. One skilled in the art is able to operatively determine optimal assay conditions for each assay by using a routine experiment.

Other steps, such as washing, stirring, filtration, and the like, can be added to the assay when the situation so requires.

One way in which an antibody of the invention can be detectably labeled is by binding the same substance to an enzyme, which is used in an enzymatic immunoassay (EIA). This enzyme, when later exposed to a suitable substrate, will in turn react with the substrate in such a way as to produce a chemical moiety that can be detected, for example, by spectrophotometric, fluorometric or visual means. Enzymes that can be used to detect labeled antibodies include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alphaglycerol phosphate dehydrogenase, triosaphosphate isomarase, horseradish peroxidase, horseradish peroxidase, horseradish peroxidase, betagalactosidase, ribonuclease, arease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine asterase. Detection can be accomplished by colorimetric methods using a chromogenic substrate suitable for the enzyme. Detection can also be performed by visual comparison of the extent of the enzymatic reaction of the substrate compared to similarly prepared standards.

Detection can be accomplished by any of a variety of immunoassays. For example, by radiolabeling antibodies or antibody fragments, it is possible to detect R-PTPase using radioimmunoassay (RIA). A good description of the RIA assay is provided in Laboratory Techniques and Biochemistry in Molecular Biology, Work, T.S. et al., North Holland Publishing Company, NY (1978), Chapter "An Introduction to Radioimmune Assay and Related Techniques, Chard, T." The radioactive isotope can be detected by such methods as using a computer or scintillation counter or autoradiography.

It is also possible to label the antibodies of the invention with a fluorescent substance. When fluorescently labeled antibodies are exposed to light at the correct wavelength, their presence can be detected by fluorescence. The most commonly used fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, pycocyanine, allophycocyanine, o-phthaldehyde and fluoresamine.

Antibodies can also be detectably labeled using fluorescent emitting metals such as ¹⁵² E or other of the lanthanide series. These metals can then be attached to the antibodies using said metal chelating groups such as diethylenetriaminepentaacetic acid (ETPA).

Antibodies can be detectably labeled by combining them with a chomoluminescent agent. The presence of cnemoluminescence-labeled antibodies is then determined by detecting the presence of luminescence that occurs during the course of the chemical reaction. Examples of particularly useful chemoluminescent markers · · · · · · · · · ·

57:.

The substances are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.

The bioluminescent agent can be used to label the antibodies of the invention. Biological luminescence is a type of chemoluminescence found in biological systems where the catalytic protein increases the efficiency of the chemoluminescence reaction. the presence of the bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent substances are labeled with luciferin, luciferase and aecvuorin for this purpose.

The antibody molecule of the invention may be engineered to be suitable for an immunometric assay, also known as a "two-way or" sandwich assay. In a typical immunometric assay, a plurality of unlabeled antibodies (or antibody fragment) bound to a solid surface or carrier and a plurality of detectable labeled soluble antibodies are added to allow detection and / or quantification of the ternary complex formed between the solid phase of the antibody, antigen and labeled antibody.

Typical and preferred immunometric assays include a forward assay in which antibodies bound to a solid support are first contacted with a test sample to extract antigen from the sample by forming a binary antibody-antigen complex. After a suitable incubation period, the solid support or carrier is washed to remove the remainder of the liquid sample comprising unreacted antigen, if present, and then contacted with a solution containing an unknown amount of labeled antibodies (which function as a reporter molecule). After the second incubation period, a complex of labeled antibodies forms with the antigen bound to the solid support or to the carrier by unlabeled antibodies. Solid substrate

5-¾. ^The carrier is washed a second time to remove unreacted labeled antibodies.

In another type of sandwich assay, where the antigen of the invention can also be used, so-called "simultaneous and" reverse assays can also be used. The temporary assay involves a single incubation step wherein the portions bind to a solid support or carrier and both labeled antibodies are added to the test sample at the same time. After incubation, the solid support or carrier is washed to remove residual liquid sample and uncomplexed labeled antibody. The presence of labeled antibodies coupled to the solid surface or to the carrier is then determined as in a conventional forward assay.

In the "reverse assay", the addition of a labeled antibody solution to the liquid sample is followed by an appropriate incubation period and the addition of unlabeled antibodies bound to a solid support or carrier. After the second incubation, the solid phase is washed in a conventional manner to remove the remainder of the test sample and the solution of unreacted labeled antibodies. The determination of labeled antibodies associated with the solid support or carrier is then determined as a "simultaneous or" forward assay.

Immunological tests

Immunological assays such as ELISA or RIA have been described in many articles, textbooks and other publications. They are described, for example, in WO 97/03998 p. 48, line 4 to p. 52, line 27. The immunoassays of the invention fall into two basic types: Immunoassays using an immobilized caspase-8 interacting protein or equivalent peptide to quantify caspase-8. Furthermore, these are immunoassays that utilize immobilized antibodies directed against the epitope of caspase-8 interacting protein to quantify caspase-8 interacting proteins.

5-9

Such assays can be used in diagnosis to determine the amount of caspase-8 and other proteins involved in the apoptotic pathway required by various disorders or syndromes arising from said pathway.

Nucleic acids

The clones obtained in the screening of the invention are considered to be partial clones. Obtaining the entire clone, if necessary, is described above. The DNA sequence of the whole clone and partial clone found in the screening of the invention has various uses.

For example, in order to manipulate the expression of caspase-8 interacting protein, it may be necessary to produce antisense RNA in the cell. Finally, all or part of the cDNA encoding the caspase-8 interacting protein is incorporated into an expression vector containing a promoter. The 3 'end of the cDNA is thereby incorporated alongside the 3' end of the promoter, wherein the 5 'end of the cDNA is separated from the 3' end of the promoter by said cDNA. Upon expression of the cDNA in the cell, an antisense RNA is generated which is unable to encode the portein. The presence of antisense RNA in the cell reduces expression of a cell (genomic) copy of the caspase-8 interacting gene.

For the production of antisense RNA, total cDNA may be used. Alternatively, a fragment thereof that contains about 9 to 2,000 nucleotides, more preferably 15 to 500 nucleotides, and most preferably 30 to 150 nucleotides, may be used.

A preferred fragment corresponds to a region at the mid 5 'end of the cDNA, more preferably a 5' end region that contains the 5 'untranslated region and / or the first exon region, and most preferably this region contains an ATG translation origin.

Alternatively, the fragment may only correspond to the DNA sequence of the 5 'untranslated region.

A synthetic oligonucleotide may be used as a nonsense oligonucleotide. Preferably, the oligonucleotide is a DNA oligonucleotide. The nonsense oligonucleotide preferably comprises between 9 to 150 nucleotides, preferably 12 to 60 nucleotides, and most preferably 15 to 50 nucleotides. The region which overlaps the nonsense oligonucleotide preferably comprises a 3 'untranslated cDNA region, more preferably it comprises a polyadenylation signal or a translation termination codon, or both.

The mechanism of action of antisense RNA and the use of antisense tools is described by Kumar et al., Microbiol. Mol. Biol. Roar. 62, p. 1415-1434, 1998 of nonsensical poligonucleotides at the potassium channel Kv1.4 dependent in Meiri et al., PNAS 95, p. 15037-15042, 1998.

The use of nonsense oligonucleotides to inhibit Bcl-x synthesis is described in Kondo et al., Oncogene 17, p. 2585-91, 1998.

The use of inhibiting gene synthesis to strain is described

The teraupeutic use of antisense drugs is described in Stix in Sci. Am. 279, p. 46, 50, 1998, Flanagan, Cancer Matassasis rev., 17, p. 169-76, 1998, Guinot end Temsamami, Pathol. Biol. (Paris) 46, pp. 347-54, 1998.

Modifying oligonucleotides that enhance properties are generally used in the design of nonsensical oligonucleotides. For example, phosphorothioate linkages are used in place of phosphorus linkages that naturally occur in DNA. This is particularly because such phosphorothioate oligonucleotides are less susceptible to degradation by cellular enzymes. Peg et al., Discloses that such unwanted in vivo side effects of phosphorothioate oligonucleotides may be possible.

reduced when a mixed phosphodiesterphosphorothioate backbone is used. Preferably, 2'-methoxyribonucleotide modifications are used in 60% of the oligonucleotides.

the oligonucleotides are capable of eliciting comparable to the effect observed by the oligonucleotides. Peng et al. Further discloses that oligonucleotide analogs that are unable to support ribonuclease H activity are not active.

Such modified antisense effect with phosphorothioate

A preferred nonsense oligonucleotide of the invention comprises a mixed phosphodiester phosphorothioate backbone. Most preferred when 2 'methoxyribonucleotide modifications are used at 30 to 80%, most preferably at 60%.

Other modifications can be introduced into the nonsense oligonucleotide. For example, the oligonucleotide molecule may bind to a group comprising a partially unsaturated aliphatic hydrocarbon chain and one or more polar or charged groups, such as a carboxylic acid group, of an ester group. Alternatively, oligonucleotides may bind to peptide structures, which are preferably membraneotrophic peptides. Such a modified oligonucleotide crosses the membrane more easily, which is critical to their function and can therefore substantially increase their activity. Membrane permeability is particularly necessary in the case of antisense drugs that must enter the brain. Palmityl-associated oligonucleotides are described in Gerster et al., Anal. Biochem. 262, p. 177-84, 1998. Oligonucleotides associated with geraniol are described in Shoji et al., J. Drug Target 5, p. 261-73, 1998. Oligonucleotides associated with peptides such as membraneotrophic peptides and their preparation are described in Soukchareun et al., Bioconjug. Chem. 9, p. 466-75, 1998. Modifications of antisense molecules or other drugs that target the molecule to certain cells and increase oligonucleotide uptake by the following:

cells are described in Wang, J. Controlled Release 53, p. 39-48, 1998.

Ribozymes

A given known mRNA gene sequence, which are ribozymes, can be designed. It is an RNA molecule that specifically binds and cleaves said mRNA sequence (see, e.g., Chen te et al., Ann. NY Acad. Sci. 660, 271-3, 1992, Zhao and Piek, Nature 365, p. 448, 1993, Shore et al., Oncogene 8, 3183, 1993, Joseph and Burke, J. Biol. Chem., 268, 24515, 1993, Shimayama et al., Nucleic Acids Symp. Ser 29, p. 177, 1993, Cantor et al. al., PNAS 90, p. 10932 (1993).

The RNA sequence encoding the ribozyme can be designed to cleave the mRNA of the caspase-8 interacting protein of the invention. The cleavage site is preferably located in the coding sequence or in the 5 'end of the non-translated region, more preferably in the 5' end of the coding region close to the initial translation codon of the AUG.

The DNA encoding the ribozyme of the invention can be introduced into cells by DNA uptake, DNA uptake (oligonucleotide and protein modifications that result in higher membrane permeability, as described below), or gene transfer mediated by a viral vector as described below. in the text.

Introduction of caspase-8 interacting proteins and their peptides and DNA into cells

The invention provides a caspase-8 interacting protein and peptides derived therefrom, DNA encoding molecules, and oligonucleotides. The therapeutic or research-related use of this tool allows them to be introduced into cells of a living organism. To this end, it is necessary to improve the permeability of peptides, proteins and oligonucleotides. Methods for increasing membrane patency for oligonucleotides are described above β3 · above. Derivatization of lipophilic structures can also be used to prepare peptides and proteins with increased membrane permeability. For example, the sequence of a known membraneotrophic peptide, as described above, may be added to a peptide or protein sequence. Further, the peptide or protein may be derivatized with partially lipophilic structures, such as the aforementioned hydrocarbon chains, which are substituted with at least one polar or charged moiety. Lauroyl derivatives of peptides are described, for example, in Muranishi et al., Pharm. Research 8, 649, 1991. Modifications of peptides and proteins further include oxidation of methionine residues to form sulfoxide groups as described by Zacharia et al., Eur. J. Pharmacol. 203, p. 353, 1991. Zacharia and co-workers disclose a peptide or derivatives thereof, wherein the relatively hydrophobic peptide bond is replaced by its ketomethylene isoester (COCH2). These and other protein or peptide modifications known in the art increase membrane patency.

An increase in membrane permeability can be achieved by using receptors, such as viral receptors, on the cell surface to induce uptake of peptides or proteins into cells. This mechanism is often used in the case of viruses that specifically bind to certain molecules on the cell surface. After binding, the cells absorb the virus into their internal environment. The cell surface molecule is called the virus receptor. These are, for example, the integrin molecules CAR and AdV and are described as viral receptors in the case of adenovirus (described in Hemmi et al., Hum. Gene Ther. 9, p. 2363-73, 1998. CD4, GPR1, GPR15 and STRL33 molecules have been identified as receptors / co-receptors suitable for HIV (see, for example, Edinger et al., Virology 249, p. 367-78, 1998).

Conjugating peptides, proteins or oligonucleotides known to bind to cell surface receptors will increase the membrane permeability of said peptides, proteins or oligonucleotides. Groups suitable for conjugate formation are sugars, vitamins, hormones , cytokines, transferrin, asiaglycoprotein, and the like Low et al., USP 5,108,921 disclose the use of these molecules to increase the membrane permeability of peptides, proteins and oligonucleotides and the preparation of said conjugates.

Low and his co-workers further disclose molecules such as folate or biotin, which can be used to target the conjugate to a number of cells in the body, due to the excessive and non-specific expression of receptors suitable for said molecules.

The above-described use of cell surface proteins to enhance the membrane permeability of a peptide, protein or oligonucleotide of the invention may also be used to target said peptide, protein or oligonucleotide of the invention to a particular cell or tissue type. For example, if it is desired to target cancer cells, it is preferred to use a cell surface protein that is expressed in large quantities on the surface of these cells. examples are the folate receptor, MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B and MUC7 mucin antigens, KSA glycoprotein antigens, carcinoembryonic antigen, prostate-specific membrane antigen (PSMA), HER-2 / neu and human choriogonadotropin beta. As described above in Wang et al., J. Controlled Release 53, p. 39-48, 1998, the use of folate for targeting cancer cells has been reported, and Zhang et al., Clin. Csncer Res. 4, p. 2669-76 1998 describes the relative excess of each of the other antigens described above for various types of cancer and normal cells.

The protein, peptide or oligonucleotide of the invention can therefore be targeted to a certain cell type as necessary using the conjugation methods described above. If it is • 9 * 9

Es:. :

For example, it is necessary to increase apoptosis in lymphatic cell cells, a protein or peptide that positively modifies caspase-8 according to the invention may be targeted to said cells using, for example, MHC class II molecules, which are expressed on said cells. This can be accomplished by coupling antibodies or antigen binding sites thereof directed against the constant region of said MHC class II molecule. A number of cell surface receptors for various cytokines and other cellular communication molecules are also described. Many of these molecules are expressed in a more or less tissue or cell restricted fashion. If the target subgroup of T cells so requires, a molecule may be used

CD4 on the surface of a T cell for producing the conjugate of the invention.

CD4 binding molecules are produced in HIV whose gp42 surface antigen is capable of specifically binding to a CD4 molecule.

The apoptosis-enhancing caspase-8 interacting protein or peptide of the invention may preferably be targeted to T cells in the treatment of a patient suffering from a T cell-based autoimmune response, such as a psoriasis patient.

Virus-mediated cellular targeting

The proteins, peptides and antisense sequences of the invention can be introduced into cells using a viral vector. The use of a vaccine vector for these purposes is described in detail in Current Protocols in Molecular Biology, Chapter 16. The use of an adenoviral vector is described in Teoh et al., Blood 92, p. 4591-4601, 1998, Narumi et al., Am. J.

Respir. Cell Mol. Biol. 19, p. 936-941, 1998, Pederson et al., J. Gastrointest Surg. 2, p. 283-91, 1998, Guang-Lin et al.,

Transplant. why. 30, p. 2923-4, 1998, Nishida et al., Spina

23, p. 2437-42, 1998, Schwarzenberger et al., J. Immunol. 161,

6383-9, 1998 and Cao et al., J. Immunol. 161, pp. 6238-44,

Retroviral transmission of antisense sequences is described in Daniel et al., J. Biomed. Sci. 5, pp. 383-94, 1998.

When viruses are used as vectors, viral surface proteins are generally used to target the virus. Many viruses, such as the adenovirus described above, are not specific to their cellular tropism. They may need to impart additional specificity to them using a promoter that is specific to a particular cell or tissue type. Griscelli et al. , Hum. Gene Therap. 9, p. 1919-28, 1998 discloses the use of a cardiac cavity-specific myosin light chain promoter 2 that is suitable for targeting a gene specifically to the heart. The transfer of said gene is carried out by means of an adenovirus.

Alternatively, the viral vector can be prepared to express another protein on its surface, or the viral vector surface protein can be altered by incorporating the desired peptide sequence. The viral vector can then be prepared to express one or more additional epitopes that can be used to target said viral vector. Cytokine epitopes, MHC class II binding peptides or epitopes derived from set molecules can be used to target a viral vector in accordance with the invention.

Application of the tools described above

The caspase-8 interacting proteins of the invention interact specifically with caspase-8 subunits that are involved in the proteolytic activity of caspase-8. Although there is no theory, it is hoped that the caspase-8 interacting proteins of the invention are elements of the lower part of the signaling pathway that includes caspase-8. The agents forming the upper portion of the signaling pathway appear to bind through the prodomain of caspase-8, which mediates the protein-protein interaction region. It appears that extensive cross-penetration of a number of agents that play a role in the apoptotic response may be mediated by protein-protein interactions that include the deadly defector region (DED) and related regions, such as those found in the predominant caspase region. In contrast to these proteins, caspase-8 interacting proteins of the invention may occur among the direct executors of cell death. For example, Tip-60, which is one of the caspase-8 interacting proteins of the invention, the histone deacetylase enzyme. The protein may therefore be directly involved in altering the chromatin structure.

The interaction of proteins of the invention with caspase-8 has several possible consequences:

1) modulation of caspase-8 activity. This is described herein in an in vivo assay wherein clone J2 of the invention inhibits caspase-8 mediated apoptosis.

2) a caspase-8 interacting protein can increase caspase-8 activity by preventing caspase-8 degradation or reducing its activity by acting as an inhibitor.

3) the activity of the caspase-8 interacting protein can be modified. This is demonstrated herein by the ability of caspase-8 to cleave proteins interacting with caspase-8. It is likely that some of these proteins are inactivated by cleavage. It is also possible that the activity of the proteins is varied by inducing new activity or that the caspase-8 interacting protein is activated by cleavage, just as caspase itself.

Consequently, caspase-8 interacting proteins, peptides, oligonucleotides and antibodies of the invention can be used to modulate caspase-8 activity. A decrease in caspase-8 activity is necessary in situations where excessive cell death occurs as a result of apoptosis. For example, it is suggested that in diseases such as multiple sclerosis with primary oligodendrogliopathy, autoimmune uveoretinitis, diabetes, psoriasis, autoimmune myocarditis I, chronic HCV-mediated hepatitis, chronic castritis such as gastritis type A, mixed connective tissue disease (MCTD), Crohn's disease colitis, body tissue damage is caused by apoptotic signals. Therefore, it may be advantageous for patients suffering from these diseases to decrease caspase-8 activity in those cells that are damaged by apoptotic cell death.

For example, in the case of the above-mentioned oligodendropathy, caspase-8 activity must be inhibited in oligodendrocytes. The cell surface protein G associated with the phospholipidlyzophosphoric acid receptor is expressed in oligodendrocytes and various other brain cells. The peptide or protein of the invention is targeted to these cells. This can be accomplished either by combining said peptide or protein with a phospholipidlyzophosphoric acid or by introducing a sequence of antibodies that specifically recognize said phospholipidlyzophosphoric acid receptor into a viral vector such that said viral vector specifically binds to a phospholipidlyzophosphoric acid receptor.

Similarly, the peptides or proteins of the invention can be targeted to other cell types involved in the aforementioned disease or disease, wherein an excess of apoptotic dead cells mediates damage to body tissue.

Also, the antisense RNA, the nonsense oligonucleotide, and the ribozyme of the invention can be targeted in a similar manner to the above-mentioned oligodendrocytes or corresponding cells of another disease. In this case, expression of caspase-interacting proteins is inhibited rather than expression of caspase-8 alone. By inhibiting the expression of a number of caspase-8 interacting proteins, the apoptotic effect of caspase-8 can be reduced. Decreasing the expression of certain caspase-8 interacting proteins can increase the effect of caspase-8, as certain proteins interacting with the ability to act as negative regulators of caspase-8 activity. The effect of using nonsense oligonucleotides and antisense RNA and ribozymes must therefore first be:: * · · · nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve nejdříve tested, for example in the above in vivo test, before such an agent is considered suitable for treatment.

On the other hand, there are certain situations where it is possible to increase caspase-8 activity. This may be the case for the same disease as described above, for example in the case of systemic lupus erythematosus disease. The cell types targeted here are different. For example, in the case of psoriasis, the population of T cells may contain autoreactive cells that are not damaged in the thymus. Therefore, the caspase-8 activity enhancing agent of the invention could be targeted to T cells. It is preferred to target the caspase-8 activity enhancer to the autoreactive cells. In some diseases such as multiple sclerosis, certain T cell clones play an important role in the development of the disease. The caspase-8 activity enhancer of the invention may therefore be targeted to such cells using one or more antibodies specifically directed to the T cell variable region of the autoreactive T cell clones, targeting the caspase-8 activity enhancer of the invention, which may be a caspase-8 interacting protein or peptide of the invention.

In accordance with the foregoing, the invention highlights pharmaceutical compositions comprising an active agent comprising one or more caspase-8 interacting proteins, peptides, antibodies, ribozymes, antisense RNA or nonsense oligonucleotides of the invention.

The invention further provides a pharmaceutical composition comprising a viral vector capable of infecting mammalian cells, said vector comprising an operably linked promoter and a DNA sequence of the invention that encodes a caspase-8 interacting protein or a peptide, ribozyme, antisense. RNA, nonsense oligonucleotide or antibodies of the invention. The viral vector may comprise a coding sequence operably linked

A promoter that encodes a peptide or protein located on the surface of a virus that is capable of binding a surface protein of mammalian cells. The surface protein is preferably a protein that allows for uptake of the viral vector and is preferably expressed in a tissue-specific or cell-type specific manner so as to allow targeting of the viral vector.

A caspase-8 interacting protein, analogs, fragments or derivatives thereof can also be used to isolate, identify and clone other proteins of the same class. That is, it binds caspase-8 or functionally related proteases or proteins involved in the intracellular signaling process. According to the invention, the aforementioned yeast two-hybrid systems can be used or a currently developed system using Souhrhr hybridization under non-stringent conditions can be used, followed by PCR cloning (Wilks et al., 1989). Wilks et al., 1989, describes the identification and cloning of two putative protein-tyrosine kinases by applying Southern hybridization under non-stringent conditions. This is followed by PCR cloning based on a known kinase motif sequence to form a kinase sequence. This approach can be used in accordance with the invention using the caspase-8 interacting protein sequence to identify and clone those caspase-8 interacting portein related proteins.

Another approach to utilizing a caspase-8 interacting protein or analogs, fragments or derivatives thereof of the invention is their use in affinity chromatography to isolate and identify other proteins or factors with which they are able to bind. They are, for example, other proteins or factors involved in the intracellular signaling process. In this application, caspase-8 interacting protein, analogs, fragments or derivatives thereof of the invention can be individually captured on affinity chromatography matrices and then come into contact with cell extracts or isolated proteins or factors believed to be involved. they participate in the intracellular signaling process. After the affinity chromatography process, other proteins or factors that bind to caspase-8 non-interacting protein or analogs, fragments or derivatives thereof of the invention can be eluted, isolated and characterized.

As mentioned above, the caspase-8 non-interacting protein or analogs thereof, fragments or derivatives of the invention can also be used as immunogens (antigens) to produce specific antibodies. These antibodies can also be used for purification of caspase-8 interacting protein (for example, human N-acetyl-glucosamine-6-phosphate deacetylase or any isoform thereof) either from cell extracts or from transformed cell lines that produce caspase-8-interacting protein or its caspase-8 protein. analogs or fragments. Furthermore, these antibodies can be used for diagnostic purposes to identify disorders related to the abnormal functioning of the FAS-R ligands or TNF system, mediating caspase-8. Such disorders may be due to a malfunction of the intracellular signaling system that includes the caspase-8 protein or caspase-8 non-interacting protein. The antibodies will serve as an important diagnostic tool.

It should also be noted that the isolation, identification and characterization of the caspase-8 interacting protein of the invention can be accomplished using any well known standard assay procedure. For example, one of such yeast two-hybrid screening methods used to identify various caspase-8 interacting proteins of the invention (in addition to various other novel proteins described above and below) is described below. above and below • •:

other methods such as affinity chromatography, DNA hybridization, etc. may be used to isolate, identify and characterize other proteins, factors, receptors, etc. that are capable of binding to caspzase-8 interacting proteins.

Overview of the drawings

Figure 1 is a diagram representing a single-chain caspase-8 construct used as bait in the two-hybrid assay described in Example 1B.

Figure 2 shows a preliminary nucleotide (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of clone 32 encoding a caspase-8 interacting protein as obtained from a cDNA clone.

Figure 3 depicts a full length putative human nucleotide sequence (SEQ ID NO: 3) and a deduced amino acid sequence (SEQ ID NO: 4) of A-acetylglucosamine-6-phosphate deacetylase which includes 5 'extension of clone J2, EST clone AA460869 and clone with exon L48741.

Figure 4A depicts the functional activity of clone J2 expressed as a percentage of apoptotic cells followed by transfection of HEK-293T cells with either p55 TNF alone (p55 TNFR) or p55TNF along with p35, a baculovirus caspase inhibitor (p55 + p35) or with clone J2 (p55 + J2) or TNF receptor p55 together with a non-cleavable mutant J2 (designated J *) that contains an Asp substitution will replace Glu at position 346 in Figure 3 (p55 + J2 *). This mutant can be cleaved either in vitro or in vivo.

Figure 4B shows the percentage of HeLane apoptotic cells alone or subsequent transfections with p35 or J2 or J2 * treated with TNF and cycloheximide.

Figure 5A shows 1725 coding pairs at the 5 'end of clone P74 (SEQ ID NO: 5).

In Figure 5B, the 574 amino acid sequence is derived from the sequence of clone P74 shown in Figure 5A (SEQ ID NO: 6).

Figure 6 is a 1428 amino acid (SEQ ID NO: 7) open reading frame obtained from the deduced amino acid sequence of the PAC clone deposited with RPCI51057120.

Figure 7 depicts the 574 amino acid sequence of the open reading frame obtained from the deduced amino acid sequence of clone p74 (designated as cloned) compared to the 1428 amino acids of the open reading frame obtained from the amino acid sequence of PAC RPCI5-1057I20 (marked as deduced).

Figure 8 shows the open reading frame arrangement of the deduced amino acid sequence of PAC clone RPCI5-1057I20 (upper sequence) (SEQ ID NO: 8) with histone deacetylase A sequence (lower sequence, GenBank, NP006028.1) (SEQ ID NO) : 9).

Figure 9A shows the autoradiograph of Bid protein and proteins encoded by cDNA clones J2 or P16 or P43 or P70 or P74 or P79 that are formed in a reticulocyte lysate in the presence of ³⁵ S methionine separated on an SDS PAGE gel.

Figure 9B shows the autoradiograph of Bid protein and proteins encoded by cDNA clones J2 or P16 or P43 or P70 or P7 4 or P7 9 produced as in Figure 9A. Proteins were examined for binding to caspase-8, which is expressed in bacteria as a fusion protein in its two subunits fused to GST. The position of the molecular weight markers is shown on the left side of the gel.

Figure 10 shows the results of cleavage of a protein encoded by a partial cDNA clone of P43 by caspase-8 or caspase-10 or caspase-3 or caspase-9 or mutants of caspase-3 or caspase-9 or caspase-10. The volumes of total bacterial lysate of recombinant caspases expressed in E. coli are reported in relative units (RU). The position of the molecular weight markers is shown on the left side of the gel. Proteins are marked with an asterisk, with the arrow with the full front portion showing the full size of the P43 protein and the arrow with the unfilled front portion showing the cleavage products.

Figure 11 shows the functional activity of proteins encoded by cDNA clones identified in a two-hybrid assay, expressed as the percentage of cells undergoing apoptosis, followed by co-transfection of HEK-293T cells with p55TNF receptor and with green fluorescent protein (labeled PC) with or without cDNA of inserts of clones J2 or P16 or P27 or P43 or P7 9 or

P74 or P70.

Figure 12 shows the activity inhibiting the death of wild-type and Tip60 non-cleavable mutant cells in HEK-293T cells co-transfected with the p55 TNF receptor and with a green fluorescent protein, as well as the effect of Δ32Tip60 lacking the first 32 amino acids of the N-terminus. Control cells were transfected with the pCGN vector alone.

DETAILED DESCRIPTION OF THE INVENTION

Example IA

Two-hybrid assay suitable for identification of caspase-8 interacting proteins.

The modified yeast two-hybrid system described as the "yeast three-hybrid system" (Tirode F. et al., J. Biol. Chem. 1997, 272 (37): 22995-9) was used to test caspase-8 interacting proteins. and its potential substrates. Single use vectors, yeast strains and libraries were obtained from Clontech (Palo Alto, USA) as components of the two-hybrid Matchmaker system (# PT1265-1).

The two caspase-8 subunits were expressed separately and their expression is under the control of different promoters. The short p10 subunit (serine at position 375 to aspartic acid at position 479) was cloned into the pGBT9 vector (Clontech) in frame with the yeast Gal4 DNA binding region (amino acids at positions 1 to 147, sequence numbering according to Laughon et al., Molecular and Cellular Biology, 4, 260-267, 1984). The long active p20 subunit (serine at position 217 to aspartic acid 374) has a mutation at position 360, i.e. cysteine at this position has been replaced by serine (C360S) conferring protease activity to the inactive enzyme. The mutated C360S p20 subunit is expressed as an unfused protein, the expression of which is driven by the Met25 promoter, which is positively regulated by methionine (Sansoda, Mol. Gen. Genet. 200, p. 407-14, 1985). The ability to control the activity of the promoter controlling expression of the p20 subunit by adjusting the concentration of methionine in a growth medium suitable for yeast cell culture, wherein 1) it is possible to use the toxic protein subunit as bait and 2) control the protein-protein interaction dependence on the third partner, p20. ' The p10 and p20 expressing units can be found on different vectors. They may also be in the same vector. The modified vector pGBT9 pGBT9.3H was used in the examples described (Tirode et al., J. Biol. Chem. 272, p. 22995-9).

1997). The p20 subunits are preferably expressed as fusions with a nucleus localized signal.

After expression, if no methionine is present, two subunits are joined in each yeast cell. Coupling of the two subunits is demonstrated by co-immunoprecipitation and Western blot experiments.

A B cell cDNA library cloned into the pGAD GH vector (Durfee et al., Genes Dev. 7, 555-569) was obtained from Dr. S. Elledge. The vector contains a Gal4 activation region (amino acids 768-881). This Gal activation region is sufficient when it fuses with the GAL4 DNA binding region to induce substantial transcriptional activity of the GAL4 gene (Ma and Ptashne, Cell, 48, 847-853 (1987)).

The upstream activation site (UAS. Sub.G) as described by Keegan et al., (1986) is present upstream of the regions of the reporter gene (lacZ) and the selectable gene (His) in the yeast strain HF7c obtained from Clontech

The HF7c culture was transformed with the above-described plasmids containing caspase-8 and the transformed yeast cells were selected based on growth on selection medium as described in the Clontech yeast culture protocols. That is, the growth selection medium does not contain tryptophan, leucine, methionine and tyosine. Homoserine was added as necessary to a concentration of 80 mg / L.

The culture of said transformants was then transformed with a vector containing a B cell library, followed by cultivation in a medium that does not contain histidine (selection medium).

• ·

Selection medium was supplemented with 3-aminotriazole, an inhibitor of the enzyme histamine synthetase. The addition of the inhibitor serves to control leakage from the promoter driving said gene in yeast cells that do not contain caspase-8 interacting proteins. In some cases, a weak non-specific interaction may lead to false transcription from the Gal4UAG-his promoter, resulting in yeast clones that grow in the selection medium.

Transformants growing in selection medium were selected and plasmid DNA was extracted. The DNA was then transformed into HB101, allowing selection in leucine-free media.

After culturing the bacteria and extracting and purifying the plasmid DNA, the plasmid DNA was then transformed into yeast SFY526 cells. The activity of the lacZ transformants SFY526 was then assayed by selection on culture medium plates containing histidine and Xgal. Yeast colonies were then transferred using Whatman 3MM # 5 filter paper (colony transfer is described in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology), then the filter paper was placed on aluminum foil for 20 seconds, transferred to liquid nitrogen, where it was left for 25 seconds to freeze the yeast cells and to allow the yeast cells to thaw at room temperature for 1 minute, then placed in a petri dish on Whatmann 3MM # 1 filter paper, previously soaked in buffer Z (a buffer suitable for reaction with beta-galactosidase), is described in Clontech protocols and in Sambrook et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, ISBN: 0879693096, Current Protocols in Molecular Biology), which contains Xgal and beta-mercaptoethanol. The colouration of blue colonies indicates active beta-galactosidase. Caspase-8 interacting proteins usually developed blue in this assay over a few minutes to overnight. It is preferred that the blue color appears within a period of 5 minutes to 3 hours, most preferably within a period of 5 minutes to one hour. Alternatively, beta-galactosidase activity was cavitated by culturing in a liquid medium containing Xgal. Cells were removed by centrifugation and the absorbance in the culture medium was measured using a spectrophotometer.

The cDNA clones identified above were further tested for interactions with other proteins. This was accomplished by transforming clones that were tested together with an irrelevant protein expressed as a fusion with the DNA activation domain in the pGAD GH vector.

Double transformants that are capable of growing in culture medium without histidine or that exhibit lacZ activity indicated that the library of clones bound non-specifically.

As well as irrelevant proteins, proteins that interact non-specifically with other proteins as well as proteins that are not related were used. For example, proteins have been tested for binding to lamin, RIP, RAIDD, TRAF2, and MORT1. In general, proteins that bind lamin are excluded.

The three hybrid assays described above were performed with the caspase-8 P10 subunit fused to the lexA DNA binding region. A preferred vector is the LexA vector available from Clontech. When using the lexA DNA binding region, the yeast strain L40 or equivalent should be used.

Caspase-8 interacting proteins were identified in the assay method described above and further analyzed using in vivo and in vitro cleavage assays and signal transduction assays as described below.

Example IB:

Modified bait for a two-hybrid assay to identify caspase-8 interacting proteins.

A modified caspase-8 bait was used in the yeast hybrid system to further test caspase-8 interacting proteins (Fields S, Song 0 Nature 1989, 340 (6230): 245-6). Caspase-8 was expressed from the pGBT9 vector (Clontech) as a single chain protein. In order to prepare a protein that could alter the conformation of the active caspase, the prodomain was removed and the small subunit 2 (starting from the cleavage at position 375 and ending with aspartic acid at position 479) was fused to the Gal4 DNA binding region. The C-terminus of the small subunit was separated from the N-terminus of the large subunit i (starting with serine at position 217 and ending with aspartic acid at position 374) with a linker containing 16 amino acids glycine to serine (GGGGSGGGGSGGGGSG). Two subunits of active caspase were then obtained from the spontaneous packaging of one molecule. Cysteine at the active site in subunit 1 was mutated with serine (sub 1 C360S). Overexpression of this single chain caspase-8 has been shown to function similarly to overexpression of wild-type caspase-8 by having the ability to induce apoptosis in HEK 293-T cells when caspase overexpression occurs from a vector similar to the pcDNAHis vector. Single chain caspase-8 activity was then blocked by co-expression of p35, a caspase inhibitor.

The two-hybrid assay was performed as described above except that the single-chain caspase-8 was expressed from the pGBT9 vector (Clontech).

Example 2: Identification of Caspase-8 Interacting Proteins

Using the modified two-hybrid assay described in Example OA, several clones were identified that encode specific caspase-8 binding proteins. Specificity • • ·

The clones were confirmed in a two-hybrid yeast assay while no binding of the control proteins was detected. Selected cDNA clones were isolated, then sequenced and the nucleotide sequences compared to those found in the Genbank database as described below.

Example 2.1

Clone L1 was found to contain a partial cDNA sequence identical to the amino acids at positions 690 to 750 Stati. Stati has been identified as a tanning factor that binds to an interferon-stimulated element (ISRE) and to a gamma-activated sequence (GAS) (Iffic SN, 1998).

State 1 has been shown to be involved in regulating the amount of constitutive caspase (Kuiner et al., 1997) and serves as an in vivo substrate for caspase-3 (King P).

et al., J. Biol. Chem. 1998, 273 (15) 8699-704). State 1 cleavage is suggested to play an important role in regulating the apoptosis response itself.

Example 2.2

Clone L7 was found to encode a partial cDNA that almost completely merges with the C-terminal part of the EST clone found in the Genbank database (accession number AA608733).

Example 2.3

Clone L20 was found to be identical to amino acids at positions 104 to 420 of NEFA (accession number 462693). NEFA is a new protein that contains a putative DNA binding region of basic amino acids with a potential nuclear signal, a region with two helix-loop-helix motifs (HLH), EF motifs, a region rich in acidic amino acids between EF motifs and a leucine zipper motif (described in Barnikol-Watanabe, S. et al., Biol Chom Hoppe Seyler 1994, 375 (8): 497-512. NEFA has been found to be a potassium binding protein and is found in the cytoplasm and on the cell surface and can be detected in the culture medium. NEFA belongs to the nucleobindine subfamily. The biological role of NEFA has not yet been fully clarified. Clone L20 is cleaved by caspase-8 in vitro and in vivo.

Example 2.4:

Clone L12 was found to encode a partial cDNA that almost completely matches the human EST clone found in the database (accession number M62097). In vitro protease assay showed that clone L12 was cleaved in vitro by caspase-8. The in vitro synthesized ³⁵ S-labeled protein was incubated for 30 to 60 minutes in protease buffer in the presence of bacterially produced caspase-8. Proteins and fragments thereof were separated into

SDS PAGE and results were visualized and phosphoimaged. Protease activity can be blocked by a specific pancapase inhibitor z-VADfluoromethylketone.

autoradiography

Example 2.5

Clone L5 was found to encode a cDNA identical to the Tip60 protein (deposit number 3024755). Tip60 (a protein of 60,000 interacting with Tat) was first described as a protein interacting with a cellular HIV-Tat transactivator (Kamine J. et al., Virol @ 1996, 216 (2): 357-66) and was later shown to exhibit histone acetyltransferase activity (Yamamoto T and Horikoshi et al., J. Biol. Chem. 1997, 272 (49): 30595-8). In addition to the ability to enhance HIV-mediated activation of the HIV promoter, the remaining biological role zbývajícíίρβΟ must be defined.

Two hybrid yeast assays revealed that clone L5 binds to caspase-8 subunit 2 alone, as well as the p10-p20 complex. Clone L5 was digested in caspase-8 in vitro by the in vitro protease assay described in Example 2.4. Tip60 protein has been cleaved in a caspase-dependent manner after »4 · 4 9

co-expression with the p55 TNF-receptor in HELa and HEK 293-T cells. After TNF treatment, it was also cleaved in HeLa cells even in the absence of protein synthesis inhibitors. The protein was cloned into the pcDNAHis vector (Invitrogen) and expressed in HEK-293-T or HeLa cells together with an apoptosis inducing protein (e.g. p55TNF-R or caspase-8). 20 hours after transfection, cells were harvested and whole cell lysate from 0.5 to 1 x 10 ⁶ cells was applied to SDS-PAGE followed by Western Blot and anti-poly-His antibodies (Sigma). Results were visualized by ECL. Several cDNA clones that encode inserts corresponding to the Tip60 protein were isolated. All of these clones including the full-length Tip60 clone (wild-type) were found not to contain a segment starting with an amino acid at position 94 (proline) to 145 (threonine). This protein section is not part of the acetylase active site and is not considered essential for protein function. It has been found that the Tip-60 mutant, wherein the aspartic acid residues at positions 200 to 203 have been replaced by alanine residues, is not cleavable.

Example 2.6

Clone M26 was found to encode a partial cDNA that almost completely corresponds to the human EST clone found in the Genbank database (accession number C18037). In the two-hybrid yeast assay, the M26 clone was found to bind to caspase-8 subunit 2 alone.

Example 3

Isolation of caspase-8 interacting proteins

Using the two-hybrid assay described in Example IB, several other clones encoding specific caspase-8 binding proteins were identified. Selected cDNA clones were isolated and sequenced, and the nucleotide sequence was compared to that found in the Genbank database as described below.

83 · «* V« * * • · · • »· 2 · 9 · · 9 ·· ··· * · * · w • · • • «1 * ·· »9 a. * • 9 9 * • * β 6 • 9 9 9 • · · V They were tested B-cell library (describing se in the publication Durfee T et al., Genes Dev. 1993, 7 (4), SSS -69) and library cell Jurkat T. In table no. 2 initial Characterization first groups of clones.

Table 2:

clone (s) part / homo log source lacZ (SFY) length inzertu cleaved several NE FA all cells + 650-1100 in vivo / in vitro B33 nucleobin din cells Ipr + 2 000 in vivo / in vitro B4.2 KIAA0615 male brain +++ 3 000 in vitro B8.1 EST00156 +++ 1 500 in vivo / in vitro Bil phosphoethanolamincytide ylyltransferase + 4- + in vitro B17.1 H23509 brain koj ence ++ 1 200 B13.1 ribonukle oziddifos fátová reductase EBV +++ 700 in vitro B22 cyclophili on 700 B27 gbAA93 635 0 + 2 700 in vivo / in vitro B37.1 EBV EBV ++++ 1 400 J2 AA746639 human ++++ 600 in vivo / in vitro J4 0 KIAA0419 male brain ++ 2 300 in vitro

in vivo: 293-T and / or HeLa cells

Partial clones encoding parts of NEFA as well as clone B8.1 were also isolated by the yeast two-hybrid method described in Example IA.

· * * * · · · · · «·« · · ««

Example 3.1

Clones B4 (accession number 3327044), B17 (accession number H23509), B27 (accession number AA936350) and J40 (accession number 2887413) encode cDNA inserts that are homologous to the end of EST.

Example 3.2

Clone B1 encodes cDNA homologs with the C-terminus of CTP phosphoenolamincytidylyltransferase (ET accession number D84307). ET is an enzyme involved in the metabolism of phospholipids and catalyzes the conversion of phosphoethanolamine to CDP-ethanolamine (Nakashima A. et al., J. Biol. Chem. 1997, 272 (14): 9567-72). Clone Bil was digested in vitro by caspase-8, as shown in the assay described in Example 2.4 above.

Example 3.3

Clones B13 and B37 encode cDNA inserts homologous to the C-terminus of the clone that encodes part of the EBV genome (accession number V01555).

Example 3.4

Clone B22 encodes a cDNA insert homologous to the C-terminus of cyclophilin T cells (accession number Y00052). Cyclophilin T cells have been identified as the intracellular cyclosporin A and FK506 receptors and exhibit intrinsic peptidylpropyl cis-trans-isomerase activity (Haendler et al., EMBO J 1987, 6 (4): 947-50).

Example 3.5

Clone B33 encodes a cDNA homolog with the C-terminus of nucleobindine (accession number 2506255). Nucleobindine is a secreted protein with DNA and Ca2 + binding ability that is very similar to the NEFA protein described in Example 3.3. Nucleobindine was originally described as a protein of 55,000 molecular weight, which increases the production of anti-DNA antibodies in cultures of mouse spleen lpt autoimmune cells (Miura K. et al., Biochem. Biophys. Res. Commun. 1992, 1787). (1), 3, 75-80). Clone B33 was digested with caspase-8 in vivo and in vitro, as demonstrated in the above assays in Examples 2.4 and 2.5.

Example 3.6

Clone J2 contains inzert o size 600 bp coding partial proteinovou sequence, on based on that arises polypeptide about molecular weight 20 May 000, if that is express e in vitro in the cell free system as well as in cells. Polypeptide encoded by the clone J2 cleaved in in vitro caspase-8 and in vivo in cells 293- -T and HeLa in common expresi s p55TNF-Rnbeo Caspase-8 ve above test

in examples 2.4 and 2.5.

The nucleotide and deduced amino acid sequence of clone J2 is shown in Figure 2.

Comparison and alignment of the 5 'end of the J2 clone by PCR with sequences published in the database showed homology of the J2 clone to human EST (accession number AA460869) corresponding to putative human N-acetylglucosamine-6-phosphate deacetylase as well as another clone (L48741). full-length human N-acetylglucosamine-6-phosphate deacetylase.

Example 4

Functional characterization of clone J2 • ·

Initial functional characterization of clone J2 showed that it exhibited caspase-8 inhibitory activity and human p55TNF-R induced apoptosis of HEK 293-T and HeLa cells. The expression of J2 suppressed / delayed apoptosis of HEK 293-T cells co-transfected with p55 TNF receptor or HeLa cells treated with TNF and cycloheximide by 25-50% is shown in Figure 3. Quantification of apoptotic cell death was accomplished by determining the portion of cells expressing beta-galoctosisase showing apoptosis morphology 20 hours after transfection of the indicated constructs. The data express the average percentage of blue-colored cells showing signs of apoptosis as part of the total number of blue cells (approximately 500 cells in the sample). Alternatively, a green fluorescent protein was used as a marker and detected by a fluorescent confocal microscope.

Example 5

Cloning of caspase-8 interacting proteins

A human placenta cDNA library expressed from the pACT2 vector (available from Clontech, Palo Alto, USA) was tested in a two-hybrid assay using the bait described above in Example IB. Using this assay procedure, several clones encoding specific caspase-8 binding proteins were identified. Furthermore, clones P16, P27, P43, P70, P74 and P79 were studied.

Sequencing of cDNA inserts of the cDNA clones P43, P16, and P74 indicated that these clones shared homologous sequences. Clone P74 contains the longest cDNA insert of approximately 3,000 bp and appears to encode a protein with a deduced open reading frame of 574 amino acids and an expected molecular weight of approximately 68,000 to 70,000. The sequences of the above three cDNA inserts were compared to sequences that were found in the published database. The cDNA inserts of the P43, P16, and P74 clones were found to have the same homology to the sequence of two EST clones in the Genbank database (W04418). and N64095). The sequences of all three cDNA inserts have been shown to contain the 3 'ends of different forms of splicing of the same protein. The sequence alignment of the three cDNA inserts confirmed the open reading frame of 574 amino acids in the P74 sequence (shown in Example 5). A similar sequence was also found in the genomic clone identified in another published database (RPCI5-1057I20, Roswell Park Cancer Institute Human PAC library), which is located on chromosome 12q31. The open reading frame derived from the sequence of said PAC clone contained 1428 amino acids (shown in Figure 6) and the above 574 amino acid open reading frame. Figure 7 shows an alignment of the open reading frame of the deduced amino acid sequence of the cDNA insert of clone P74 (labeled "cloned") with the open reading frame deduced from the sequence of the PAC clone (labeled "deduced").

Comparing the deduced amino acid sequence of the P74 clone and the sequence deduced from the full-length PAC clone, it is clear that the full-length protein corresponding to P74 is longer at the 5 'end and may likely start with 6.

It has been found that the sequence of clone P74 also exhibits substantial homology with the highly homologous region of mice and human histone deacetylase, a region that may contain the anzymatic active site of histone deacylase. This suggests that the protein encoded by P74 may share the function of said proteins. The sequence in the region between 163-1776 bp of the partial cDNA of clone P74 shows approximately 80% homology to histone deacetylase A (accession number NP-006028). The sequence in the region between 385-1707 bp of the partial cDNA of clone P74 flanges by homologization of histone deacetylase 5 (accession number NP-005465). A sequence in the region between 418-1629 bp of the partial cDNA of clone P74 shows homology to the histone deacetylase mHDA sequence (accession number AAD09834) and a region sequence between 424-1551 bp of the partial cDNA of clone P74 shows homology to the histone deacetylase 6 sequences (accession number NP-) 006035). Figure 8 shows a comparison of the amino acid sequence of the full-length protein deduced from the PAC sequence with the histone deacetylase A sequence (GenBank Accession No. NP-006028.1).

Example 6

Functional characterization of caspase-8 proteins

(A) Proteins encoded by cDNAs of clones J2 or P17 or P43 or

P70 or P74 or P79, which were identified by a two-hybrid assay, were expressed in a reticulocyte lysate in the presence of 35S-labeled methionone in a TnT T7 coupled rethylococyte lysate system (available from promega, Cat # L4610) and separated on SDS-PAGE gel ( shown in Figure 9A). The same amount of proteins expressed in reticulocyte lysates were analyzed for the ability to bind the two caspase-8 subunits GST-S2-S1 (C360S) described in Example IB above with GST and the ability to express in bacteria. After pre-purification for 1 hour incubation at 4 ° C with GST TnT particles alone, the proteins produced precipitated with GST-S2-S1 caspase-8 fusion protein associated with GST particles during incubation with GST particles for 1 hour at 4 ° C. The GST precipitate was washed and the caspase-8 bound proteins were separated from the particles by coating in SDS-containing sample buffer and resolved on SDS-PAGE. Proteins capable of binding to the caspase-8 construct could then be visualized by autoradiography. The protein encoded by clone P74 has been shown to specifically bind in vitro to caspase-8 (Figure 9B) as compared to the binding of GST-S2-S1 β;

(C360S) with Bid, a known proximal substrate of caspase-8 on the Fas apoptosis signaling pathway. The full-size proteins produced in the reticulocyte lysate are marked with asterisks in the figure.

In the two-hybrid assays described in Example IA above, it was found that the protein encoded by clone P74 binds to a fusion protein encoded by caspase-8 subunits, but does not bind to a small caspase8 subunit that is expressed alone. For comparison, other proteins exemplified above in examples such as Tip60 bind to the small subunit of S2 caspase-8 when expressed alone (data not shown).

The protein encoded by the cDNA of clone P43 was expressed in vitro in a reticulocyte lysate in the presence of 35S methionine using the TnT system T7-linked reticulocyte lysate and subjected to caspase-3 cleavage of recombinant wild-type or mutant thereof or caspase-9 or caspase-10 expressed in as fusion proteins, histidine-labeled 2-subunit 1 (S2-S1) subunit. Caspase-8 was expressed as histidine-labeled subunit 2-subunit 1 (S2-S1) as fusion proteins. 1 and 4 or 4 (Figure no.

total volumes defined as (RU) were used 10). In vitro bacterial lysate, relative units 1 in the protease assay, synthesized 35S-labeled proteins were incubated for 30 minutes in the rotease buffer at 37 ° C in the presence of biotterially produced caspase-8. Proteins and fragments thereof were separated by SDS-PAGE and the results were visualized by autoradiography or phosphoimaging. The results indicate that the protein encoded by the P43 cDNA clone used as a substrate was efficiently cleaved by caspase-8, weakly cleaved by caspase-10, and not cleaved by caspase-3 or caspase-9 (shown in Figure 10). The protein appears to be a specific substrate for caspase-8.

C) In order to analyze the effect of the newly cloned proteins on the apoptosis cell death induced by the TNF receptor signaling pathway, the selected cDNA was cloned into pcDNA 3.1 / His C expression vectors (available from Invitrogen) and temporarily cotransfected with the TNF receptor p55 expressed in vector pcDNA3 fluorescent with green expressed (Invitrogen) and protein (GFP) from the pEGFPCl expression vector (Clontech) into HEK-293-T cells. Tip60 cDNA and Δ32-Τίρ60 lacking the first 32 N-terminal amino acids were cloned into the pCGN vector (described in M. Tanak and where

W. Herr, Cell 60, 375-386, 1990), a hemagglutinin tag (HA) fused to the N-terminus of cDNA and used in the same experiment.

After 24 hours, the transfected cells were tested with a fluorescence microscope and cell death was determined by determining the number of cells that exhibit apoptosis morphology of the total fluorescent cell population. Overexpressed P74 protein from a partial cDNA cloned into the pcDNA-His vector was found to protect HEK-293-T and HeLa cells from death caused by overexpression of the TNF p55 receptor (shown in Figure 11) or overexpression of fused two caspase- subunits. 8 (data not shown). Wild-type ΤίρβΟ and a cleavable mutant Tip60 where the aspartic acid residues at positions 200 and 203 were replaced with alanine residues were found to protect HEK-293-T cells from death caused by overexpression of the p55 TNF receptor (Figure 12), while A32 -Tip60, which does not contain the first 32 N-terminal amino acids, does not show a protective effect.

• ·

Based on the described invention, one of ordinary skill in the art can achieve the same results over a wide range of equivalent parameters, concentrations, and conditions without departing from the scope of the invention and must conduct experiments.

(2) Information about SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 447 pairs of cows (B) TYPE: nucleic acid (C) CHAIN TYPE: (D) TOPOLOGY: (ii) SPECIES MOLECULES: DNA (Vi) ORIGINAL SOURCE: (AND) ORGANISM: Homo sapiens (Xi) Sequence Description: SEQ ID NO: 1:

cggcacgagg gcctgggcaa cggccggcac acgctgggac agcaggaagt ggaagtggac 60 ggtctgacgg cctacgtggc aggtgagcgc cctgacccac tgggtcccag gtcccagccc 120 gcatgccagg tggcccacga cccccccaga gcctgccctc tctgctctca aggcaccaag 180 acgctgagtg gcagcatagc cccaatgaac gtctgtgtcc gggcacttcc tgcaggccac 240 aggttcagca tgaagtcggc cttgaaggct gcatccttgc accccgccca gttgctgggg 300 ctggagaaga gtaaggggac cttgactttg gtgctgaegc agacttcgtg gtgctcgacg 360 actcccttca cgtccaggcc acctacatct cgggtgagct ggtgtggcag gcggacgcag 420 ctaggcagtg acaaggacct cggctga 447 (2) Information about SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 148 residues (B) TYPE: amino acid (C) CHAIN TYPE: (D) TOPOLOGY: (ii) SPECIES MOLECULES: protein (vi) ORIGINAL SOURCE: (AND) ORGANISM: Homo sapiens

(xi) Sequence description: SEQ ID NO: 2:

Arg Kis Glu Leu Gly

Asn Gly His His Thr Leu Gly Gin Gin G1 10 15 i

• · · · 4 · 4 4 4 4 * * * * * * * * * * * * * * * * * * * * *

Wall Glu Wall Asp 20 May Gly Leu Thr Ala Tyr 25 Wall Ala Gly Glu A.rg 30 For Asp For Leu Gly For Arg c e r Gin For Ala Cys Gin Wall Ala Kis Asp For 35 40 45 For Arg Ala Cys For Leu Cys Ser Gin Gly Thr Lys Thr Leu Ser Gly 50 55 60 Ser Ile Ala For Met Asn Wall Cys Wall Arg Ala Leu For Ala Gly His 65 i C 75 80 Arg Phe Ser Met LV5 Ser Ala Leu Lys Ala Ala Ser Leu His For Aia O 90 95 Gin Leu Leu Gly Lee Glu Lys Ser Lys Gly Thr Leu Thr Leu Wall Leu 100 ALIGN! 105 110 Thr Gin Thr Ser Trp Cys Ser Thr Thr For Phe Thr Ser Arg For For 115 120 125 Thr Ser Arg Wall Ser Trp Cys Gly Arg Arg Thr Gin Leu Gly Ser Asp

130

Lys Asp Leu Gly 145 (2) Information about SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 1 513 pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: (D) TOPOLOGY: SPECIES MOLECULES: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (xi) Description of sequence: SEQ ID NO: 3:

ggcgcggctc cgctctcggc tggggttcgt • · · cactgggcgc • * · ··· ·· gggatttggc • · · · * cgccgcg§ £ fg 60 ctccggagcc gctcgctccc gacacggctc acgatgcgcg gcgacagggc cggcgggggc 120 cccgtgcrcc agttcactaa ctgccggatc ctgcgcggag ggaaactgct cagggaggat 180 ctgtggg-gc gcggaggccg catcttggac ccagagaagc tgttctttga ggagcggcgc 240 ' gtggccgacg agcggcggga ctgcgggggc cgcatcttgg ctcccggatt catcgacgtg 300 cagatcaacc gtggatttgg tgttgacttc tctcaagcca cggaggacgt ggcttcgggg 360 qttqccczcg tggcccggag gatcctgrcg cacggcgtca cctccttctg ccccaccctg 420 gtcacttccc caccggaggc ttatcacaag gttgttcctc agatccctgt gaagagtggt 480 ggtccccatg gggcaggggt cctcgggctg cacctggagg gccccttcat cagccgggag 540 aagcggggcg cgcaccccga ggcccacctc cgctccttcg aggccgatgc cttccaggac 600 ttgctggcca cctacgggcc cctggacaat gtccgcatcg tgacgctggc cccacagttg 660 ggccgtagcc acgaagtgat ccgggcgctg acggcccgtg gcatctgcgt gtccctaggg 720 cacccagtgg ctgacctgcg ggcggcagag gatgctgtgt ggagcggagc caccttcatc 7S0 acccacc: ct tcaacgccat gctgcctttc caccaccgcg acccaggcat cgtggggctc 840 ctgaccagcg accggctgcc cgcaggccgc tgcatcttct atgggatga ” tgcagatggc 900 acgcacacca accccgccgc cctgcggatc gcccaccgtg cccatcccca ggggctggtg 960 ctggtcaccg atgccatccc tgccttgggc ctgggcaacg gccggcacac gctgggacag 1020 cagaaagtgg aagtggacgg tctgacggcc tacgtggcag grgagcgccc tgacccactg 1080 ggtcccaggt cccagcccgc argccaggtg gcccacgacc cccccagagc ctgccctctc 1140 tgctctcaag gcaccaagac gctgag-ggc agcatagccc caatgaacgt ctgtatccgg 1200 cacttcctgc aggccacagg ctgcagcatg gagtcggccc tggaggctgc atccctgcac 1260 cccgcccagt tgctggggct ggagaagagt aaggggaccc tggactrtgg tgctgacgca 1320 gacttcgtgg tgctcgacga ctcccttcac gtccaggcca CCČčtCcí cxc gggtgagctg 1330 gtgtggcagg cggacgcagc taggcagtga caaggacctc ggcrgagagg acacctggcc 1440 gcagcgggat gccatcaggg ccgggtggtt ggggagctgg tctccaggga gtgagtcggg 1500 agccctgctg gat 1513 Information about SEQ ID NO : 4:

SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 502 residues (B) TYPE: amino acid (C) CHAIN TYPE: (D) TOPOLOGY: SPECIES MOLECULES: protein

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(xi) Gly T • X Description Ala Ala sequence: SEQ Ala ID Gly NO: Wall 4 Ala Arg Asp 15 Dec Leu For Leu 5 Ser Arg 10 His Trp Ala Ala A-š Gly 20 May Leu Arg Ser Arg Ser 25 Leu For' Thr Arg Leu 30 Thr Met Arg Gly Asp Arg Ala Gly Gly Gly 40 For Wall Leu Gin Phe 4 5 Thr Asn Cys Arg Ile Leu 50 Arg Gly Gly Lys 55 Leu Leu Arg Glu Asp 60 Leu Trp Wall Arg

• 4 4 · · · ·

95 < 4 • • 4 4 • 4 • • • 4 4 • 4 4 · · · 4 4 4 4 4 4 Gly Glv Arg Ile Leu Asp For Glu Lys Leu Phe Phe Glu Glu Arg Arg 65 70 75 80 Wall Ala Asp Glu Arg Arg Asp Cys Gly Gly Arg Ile Leu Ala For Gly 85 90 95 Phe Ile Asp Wall Gin Ile Asn Arg Gly Phe Gly Wall Asp Phe Ser Gin 100 ALIGN! 105 110 Ala Thr Glu Asp Val Gly Ser Gly Wall Ala Leu Wall Ala Arg Arg Ile 115 120 125 Leu Ser His Gly Val Thr Ser Phe Cys For Thr Leu Wall Thr Ser For 130 135 140 For Glu Ala Tyr. HisLys Wall Wall For Gin Ile For Wall Lys Ser Gly 145 150 155 160 Gly For His Gly Ala Gly Wall Leu Gly Leu His Leu Glu Gly- For Phe 165 170 175 Ile Ser Arg Glu Lys Arg Gly Ala His For Glu Ala His Leu Arg Ser iao 18 5 190 Phe Glu Ala Asp Ala Phe Gin Asp Leu Leu Ala Thr Tyr Gly For Leu 195 200 205 Asp Asn Wall Arg Ile Val Thr Leu Ala For Glu Leu Gly Arg Ser His 210 215 220 Glu Wall Ile Arg Ala Leu Thr Ala Arg Glv Ile Cys Wall Ser Leu Gly 225 230 235 240 His Ser Wall Ala Asp Leu Arg Ala Ala Glu Asp Ala 'Wall Trp Ser Gly 245 250 255 Ala φ V-, y Phe Ile Thr Krs Leu Phe .Asn Ala Met Leu Prc Phe His His 260 265 270 Arg Asp For Gly Ile Val Gly Ls u Leu y— Ser Asp Arg Leu For Ala 275 280 285 Gly Arg Cys Ile Phe Tyr Gly Met Ile Ala Asp Gly Thr His Thr Asn 290 295 300 For Ala Ala Leu Arg Ile Ala His Arg Ala His For Gin Gly Leu Wall 305 310 315 320 Leu Wall Thr Asp Ala Ile For Ala Leu Gly Leu Gly Asn Gly Arg His

(+420) 325 330 335

Thr Leu Gly Gin Glu Glu Glu Val Asp Gly Leu Thr Ala Tyr Val 340 345 350

Ala Gly Glu 355 Arg Pro Asp Pro Leu 360 Gly For Arg Ser Gin 365 For Ala Cys Gin Val Ala 370 His Asp For Pro Arg 375 Ala Cys For Leu 380 Cys Ser Gin Gly Thr 385 Lys Thr Leu Ser Gly Ser Ile 390 Ala For Met 395 Asn Wall Cys Wall Arg 400 His Phe Leu Gin Ala 405 Thr Gly Cys Ser Met 410 Glu Ser Ala Leu Glu 415 Ala Ala Ser Leu His Pro 420 Ala Gin Leu Leu 425 Gly Leu Glu Lys Ser 430 j-iys Gly Thr Leu Asp 435 Phe Gly Ala Asp Ala 40 Asp Phe Wall Wall Leu 4 4 5 Asp As ρ Ser Leu Hus Val , i ~ n * 1 v_ / Gin Ala Thr Tyr Ile 455 Ser Gly Ό -L U Leu 460 Trp Gin Ala Asp 465 Ala Ala Arg Gin Gin Gly Pro 470 Arg Leu Arg 475 Gly His Leu Ala Ala 4 80 Al a Gly Cys His Gin 485 Gly Arg Val Wall Gly 490 C-iu Leu Wall Ser Arg 495 Glu Wall Gly Ser Pro Ala Gly

500 (2) Information about SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 1,725 pairs of flanks (B) TYPE: nucleic acid (C) CHAIN TYPE: (D) TOPOLOGY: SPECIES MOLECULES: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (xi) Description of sequence: SEQ ID NO: 5:

gagcagctca cggctgcggc gtggtggacg cccgctcctc cggctccccc cggcctctgt cctgccagcc accacagggc agcaggcacc gggctccgga tcggtccact ctggacaacg ggtggggttg cgctgggccg aatggtttcg ttctgcttct agcaagatcc taccaagacc ccagggagtg gtggcctggg aggatagtcg ggatrtgatg tgttttggat ttggagggtg cttctgggta aattccatcc cagcgcctgg gaagtgaagg ccctcagagc aaactcacgt agataccctc atggcctgga tccagcagca ggggcagcac cccgggctca aggcccgagt tgatctatga cggagcacgc gccagtgtga ctgagcggca ggaagctggc gggtggacac ctggcagtgt ctgtggtgcg tcaactcagt tcattgtaga ccagtgtgct gggctgtgga ctggaggtct tgatgcccat ctgctgaggg acatgacgca gccatgacct acagggtgga gctctctgga cctcctgtcc caataaccgc agctggtgga ccaggtgatc ggctgaagac gcacagggag ccctcaggtg cggggacact gtcttcccca cctctccagc ctcggtcatg cggccgcatc gtgtctccga cgtgctcctc agggctcctg tgacaccatc cactgacctc gcccccagga ggccatcgcc ctgggacgtg ctacatctcc tgaggtagga ggaccccccc cgcccgagag tcacccggcc gcaactgatg cacagccatc tcccctttca ggccgtgatc agactcctgg actggcgtcc ggaggaagaa aagaggtcag ctggagacag ctgggccatg ttgctctggg gtgctgcttc gccgcacctg tcagagaccc ctgaagcacc cagagcatct ggccggaagg tacggcacca gcacagcgga tggaatgagc gccttcaaag caccatgcag tgccggcagc caccatggca ctgcatcgcc gctggcagcg atgggggatc ttctctccag ccactgggtg aacctggcag tgtgacgcct gaagaaggct cgggtgcaca gtgcctagag ctctctgtgg cctatgaatc ccaagccgag atggcggggg ggcagcctga aacagcagcg ctctggccca cctcactgtc ctgccaggac agtgctcctg ggtcccggct cctccctgga acccgctcag tgtttgtgat ttcattcctc tggct tcccg atcattcaac tgcaacagca acggcaccca atgacgacgg gtgagggctt ctgagtacct acctagtcct gctaccatgt gaggcgcagt ctgaggcccg ggaaacagaa gtaaatactg tgccaggggc gcatcctggc tctaa tgagaagccc accgggccag ggccagaggc actggctggg gggtgggcac agccccagag cctgcccttc cggtgacaac gcaggagcgg agagctgcag ccgcctcaaa gctgccctgt caatgcagcc tgagctaaag agccatgggc gagcaaggcc gcaaaccttc caacttcttc caatgtcaat ggctgctttc ggtgtctgct ttctgccaaa ggtgctggcc tgtggctgct acccaacctc gggctgcatg tgacaaagaa tgaagatagg

120

180

240

300

360

420

480

540

600

660

720

780

840

900

960

1020

1080

1140

1200

1260

1320

1380 1440 1500 15 60 1620 1680 1725 (2) Information of SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(AND) LENGTH: 57 4 'residues (B) TYPE: amino acid (C) CHAIN TYPE: (D) TOPOLOGY: (ii) SPECIES MOLECULES: protein (vi) ORIGINAL SOURCE: (AND) ORGANISM: Homo p

(xi) Sequence description: SEQ ID NO: 6:

·· · · · · · · · · · · · · · · · · · · · · ·

Glu 1 C-ln Leu Lys Thr 5 His Wall Gin Wall Ile 10 Lys • · Arg «· ♦ · Ser • · Ala • · · Lys 15 Dec * For Ser Glu Lys For Arg Leu Arg Gin Tle For Ser Ala Glu Asp Leu Glu 20 May 25 30 Thr Asp Gly Gly Gly For Gly Gin Wall Wall Asp Asp Gly Leu Glu His 35 40 45 Arg Glu Leu Gly His Gly Gin For Glu Ala Arg Gly For Ala For Leu 50 55 60 Gin Gin His For Gin Wall· Leu Leu Trp Glu Gin Gin Arg Leu Ala Gly 65 70 75 80 Arg Leu For Arg Gly Ser Thr Gly Asp Thr Wall Leu Leu For Leu Ala 85 90 95 Gin Gly Gly His A.rg For Leu Ser Arg Ala Gin Ser Ser For Ala Ala 100 ALIGN! 105 110 For Ala Ser Leu Ser Ala For Glu For Ala Ser Gin Ala Arg Wall Leu 115 120 125 Ser Ser Ser Glu Thr For Al a Arg Thr Leu For Phe Thr Thr Gly Leu 130 135 14 0 Ile iyr Asp Ser Wall Met Leu Lys His Gin Cys Ser Cys Gly Asp Asn 145 150 155 160 Ser Arg His For Glu His Ala Gly Arg Ile Gin Ser Ile Trp Ser Arg 165 170 175

Leu Gin Arg Gly Leu Arg Ser Gin Cys Glu Cys Leu Arg Gly Arg

180 185 190

Lys Ala Ser Glu Glu Glu Ser Glu Ser Glu Ser Glu Arg His Val 195 200 205

Leu Leu Tyr Gly Thr Asn For Leu Ser Leu Lys Leu Asp Asn Gly 210 215 220

Lys Leu Gla Leu Leu Gin Arg Met Phe Val Leu Pro Cys

(+420) 225 230 235 240

Gly Val Gly Val Asp Thr Asp Thr Ile Trp Asn Glu Leu His Ser

245 250 255

Ser Asn Ala Ala Arg Trp Ala Ala Gly Ser Thr Asp Leu Ala Phe 260 265 270

Lys Val Ala Ser.Arg Glu Leu Lys Asn Gly Ph

Gly His His Ala Asp His Ser Thr Ala Met Gly Phe Cys Phe Phe 290 295 300

Asn Ser Val Ala Ile Cys Arg Gin Leu Gin Gin Gin Ser Lys Ala

305 _ 310 315 320

Ser Lys Ile Leu Ile Val His Trly His His Gly Asn Gly Thr

(+420) 325 330 335

Gin Gin Thr Phe Tyr Gin Asp Ser Ser Leu His 340 345 350

Arg His Asp Gly Asn Phe Phe Pro Gly Ser Gly 355 355 365 365

Val Gly Ala Gly Ser Gly Glu Phe Asn Val Asn Val Ala Trp Ala 370 375 380

Giv Gly Leu Asp Pro Met Gly Asp Pro Glu Tyr Leu Ala Ala Phe

385 390 395 400

Arg Ile Val Val Met Pro Ile Ala Arg Glu Phe Ser Pro Asp Leu Val

405 410 415

Leu Val Ser Ala Gly Phe Asp Ala Ala Glu His Pro Ala Pro Leu 420 425 430

Gly Gly Gin Gly Tyr His Val Ser Ala Lys Cys Phe

100 · r r r · · · t t t t t t t t t t t t t t t t t ·· * «

435 440 445

Leu Met Asn Leu Ala Gly Gly Val Val Leu Ala Leu Glu Gly 450 455 460

Kis Asp Leu Thr Ala

465,470 4 ~ 5,430

Leu Glu Gly Asn Arg Val Asp For Leu Glu Glu Glu Glu Tro Lys Gin

485 490 495

Lys Pro Asn Leu Asn Ser Iu Arg Ser Iu Arg Glu Ala Val Ile Arg Val 500 505 510

Kis Ser Lys Tvr Trp Gly Cys Met Gin A.rg Len Ala Ser Cys Pro Aso 515 520 525

Ser Trp Val Pro Glu Ala Asp Lys Glu Val Glu Ala 530 535 540

Val Thr Ala Leu Ala Glu Ile Leu Ala Glu Asp Arg 545 550 555 560

Pro Glu Gin Glu Glu Glu Glu Glu Glu Pro Glu Glu 565 570 (2) Information about SEQ ID NO: 7:

(and) CHARACTERISTICS SEQUENCE: (A) LENGTH: 1,428 residues (B) TYPE: amino acid (ii) MOLECULAR TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM : Homo sapiens (Xi) Sequence description: SEQ ID NO: 7: Met Phe Ala Arg Ser Ala Gly Leu Cys Phe Trp Val Gly Val 1 5 10 15 Ser His Gly Gly Asp Ala Glu Glu Val Leu Gin His Pro Thr Pro 20 May 25 30 Thr Gly Arg Arg G Pro Arg Pro Pro Ser Ser Ala 35 40 45 Glu Gly Asp For Gly Met Leu Lys Cys Gly Cys Val Pro Ser Pro 50 55 60 Gin Lys Val Ala Leu Val Gly Ala Pro Phe Cys

9

101

Phe Gin Arg Phe Gle Gin Gy Giy Ser

90 95

For Glu Ser Ala Arg For Arg Asn Arg Gin For Tyr Ala Thr Gin Asn 100 105 110

Gly Pro Ala Pro Arg Pro Gin Val Leu Pro Gly Ser Ser Ser Arg Cys 115 120 125

Cys His Gly Tyr Ile Cys Phe Leu Phe Ser Ser Gin Thr A.Ia Glu ± 30 135 140

Val Glu Val Gly Trp Gly Gly Asp Thr Gly Ser Gin Leu Arg Pro Leu

145 150 ± 55 ± 60

Leu Arg Gly Ala Val Tyr As Ser Ser Trp Asp Ser Gin Lys Glu

165 170 175

Asp Ser Lys Pro Ile Leu Arg Leu Gin Asn Thr Gin Leu Phe His 130 185 190

Ser Val Ser Leu Ser Gr Thr Gin Val Ser Pro Gly A.la Kis 195 200 205

Tyr Cys Pro Pro Pro Gly Ala Gly Pro Pro Pro Pro Pro Ala A.sp Thr 210 215 220

Pro Gly Pro Gin Pro Gin Pro Pro G Asp Pro Le G Arg Pro Gly Gin Pro Arg

(+420) 225 230 235 240

Pro Glu Pro Pro Glu Pro Pro Thr Leu Leu Ala Leu Gin A.rg Pro

245 250 255

Gin Arg Leu Gin Gin Arg 260 265 270

Ser Val Glu For Met Arg Val Lys For Glu Leu Pro Ala Cys Gly -Ala 275 230 285

Thr Leu Ser Leu Val Le Ser Pro Le Phe Ser Ile.Prc Arg His 290 295 300

Gin Ser Gin Ser Ser Thr Pro Cys Pro Phe Leu Gly Cys Arg Pro Cys 305 310 315 320

Pro Gin Leu Ser Met Asp Thr Pro Met Pro Glu Leu Gin Val Gly Pro 325 330 335

Lys Asp Lys

C-1 Glu Gln

Ser Lys Glu 355

For Al Ala 370

Gin Lys Leu 335

Arg Thr Val

Pro Cys Ser

For Trp His 435

Ala Thr Arg 450

For Ser Asp 465

For Asn Leu

Lys Asn Pro

Arg Pro Ala 516

For Ala Ser 530

For Lie Leu 54 5

Glu Thr Ser

102

Glu Leu Arg Gin Leu Leu His 340 345

Val Ala Thr Pro

Ala Cys Val Cys Ala Val 375

Ala Glu Val Ile Leu Lys 390

His Pro Asn Ser Pro Gly Ile 405 410

Gly Gin Pro Cys Ser Val 420 425

Ser Phe Cys Arg Thr Leu Glu 440

Ser Met Leu

Pro Glu His Phe Pro Leu 470

Lys Leu Arg Lys Pro Lys 485 490

Leu Leu Arg Lys Glu Ser Ala 500

Glu Thr Leu Gly Asp Ser Ser

Gly Cys Ser Ser Pro Asn Asp 534

Gly Ser Glu Ala Leu Leu Gly 550

Val Ala Pro Phe Ala Leu 565 570

Ser Pro Thr 365

Ala Ser Ser 380

Gin Gin Ala

For Tyr Arg

Pro Thr Pro

For Leu Glu 445

Pro Pro Val

460 · · • • • • • • • • •

• * »· · · ·

Ser Lys Arg 350

Ser Gin Val

Val Lys

Ala Leu Glu 400

Ser Gin Gly

Leu Lys Gin

Thr Glu Gly

For Ser Leu

Arg Lys Thr

475 480

Lys Ser Leu Arg Arg 495

For Pro Le Ser Arg Arg 510

Pro Ser Ser Ser Ser Thr 525

Ser Glu His Gly Pro Asn 540

Gin Arg Leu 555 560

Thr Val Ser Leu Le 575

Ala Ile Thr Leu Gly Leu

103 • · • • • • • · • · • · • • · • · • · · • • • • • • · • • • • · · • • · • • · • • · · • · • · • · • · • · • · • · »* • « • · • · • · Arg Thr His For Thr Leu Gly For Arg Gly For Ile Leu Gly Ser For 595 600 605 His Thr For Leu Phe Leu For His Gly Leu Glu For Glu Ala Gly Gly 610 615 620 Thr Leu For Ser Arg Leu Gin For Ile Leu Leu Leu Asp For Ser Gly 625 630 635 640 Ser His Ala For Leu Leu Thr Wall For Gly Leu Gly For Leu For Phe 645 650 655 His Phe Ala Gin Ser Leu Met Thr Thr Glu Arg Leu Ser Gly Ser Gly 660 665 670 Leu His Trp For Leu Ser Arg Thr Arg Ser Glu For Leu For For Ser 675 680 685 Ala Thr Ala For For For For Gly For Met Gin For Arg Leu Glu Gin 690 695 700 Leu Lys Thr His Wall Gin Wall Ile Lys Arg Ser Ala Lys For Ser Glu 705 710 715 720 Lys For Arg Leu Arg Gin Ile For Ser Ala Glu Asp Leu Glu Thr Asp 725 730 735 Gly Gly Gly For Gly Gin Wall Wall Asp Asp Gly Leu Glu His Arg Glu 740 745 750 Leu Gly His Gly Gin For Glu Ala Arg Gly For Ala For Leu C-ln Gin 755 760 765 His For Gin Wall Leu Leu Trp Glu Gin Gin Arg Leu Ala Gly Arg Leu 770 775 780 For Arg Gly Ser Thr Gly Asp Thr Wall Leu Leu For Leu Ala Gin Gly 785 790 795 800 Gly His Arg For Leu Ser Arg Ala Gin Se Γ- Ser For .Ala Ai a For Ala 805 δΙΟ 815 Ser Leu Ser Ala For Glu For Ala Ser Gin Ala Arg Wall Leu Ser Ser

820 825 830

Ser Glu Thr Al Ala Thr Leu Phe Thr Thr Gly Leu Ile Tyr 835 840 845 • ·

104 · · · · · · · · · ·

Asp Ser 850 Wall Met Leu Lys His 855 Gin Cys • Ser • · • · · · · · • • · · Asp • • · · Asn • • · Ser • · • · Arg Cys Gly 860 His For Glu His Ala Gly A.rg lie Gin Ser Ile Trp Ser Arg Leu Gin 865 870 875 880 Glu A_c Gly Leu Arg Ser Gin Cys Glu Cys Leu Arg Gly Arg Lys Ala 885 890 895 Ser Leu Glu Glu Leu Gin Ser Wall His Ser Glu Arg His Wall Leu Leu 900 905 910 Tyr Gly Thr Asn For Leu Ser Arg Leu Lys Leu Asp Asn Gly Lys Leu 915 920 925 Ala Gly Leu Leu Ala Gin Arg Me t Phe Wall Met Leu For Cys Gly Gly 930 935 940 Wall Gly For Leu Ala Thr Leu Ser Al a Phe Leu Ala Ser Leu Ala For 945 950 955 960 Thr Wall For Gin Gly Leu Ser Arg Wall Ser Trp Gly Leu Lys For For 965 970 975 For Gly For Asn For Lys Ser Arg For ALa For Cys For Trp Gly For 980 985 990 Gly Arg Gly Wall Gly Thr Thr For Leu Gly For Gly Ser Cys Wall Lys 995 1000 1005 For Trp Met Met Arg Ala Leu Thr Leu Ala For Gin Wall Asp Thr Asp 1010 J 015 1020 Thr Ile Trp Asn Glu Leu His Ser Ser Asn Ala Ala Arg Trp Ala Ala 1025 1030 1035 1040 Gly Ser Wall Thr Asp Leu Ala Phe Lys Wall Ala Ser Arg Glu Leu Lys 1045 1050 1055 Asn Gly Phe Ala Wall Wall Arg For For Gly His His Ala Asp His Ser 1060 1065 1070 Thr Ala Met Gly Phe Cys Phe Phe Asn Ser Wall Ala Ile Ala Cys Arg 1075 1080 1085

Gin Leu Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin

105

105 • • • · · • • · • · · · • • • • · • • · • • • · · • • · • • • · · • · • • • · • · * · • · • · • · Asp Val His Kis Gly Thr Gin Gin Thr Phe Tyr Gin Asp For 1105 1110 1115 1120 Ser Val Leu His Arg His Asp Asp Gly Asn Phe Phe 1125 1130 1135 For Gly Ser Gly Ala Val Asp Glu Val Gly Ala Gly Ser Gly Glu Gly 114 0 1145 1150 Phe Asn Val Ala Gly Leu Asp For For Met Gly 1155 1160 1165 Asp Pro Glu Tyr Leu Ala Ala Phe Arg Ile Wall Wall Met For Ile Ala 1170 1175 .180 Arg Glu Phe Ser For Asp Leu Val Leu Ser Ala Gly Phe Asp Ala 1185 1190 1195 1200 Ala Glu Gly His Pro Gly Gly Tyr His Wall Ser Ala Lys 1205 1210 1215 Cys Phe Gly Gin Leu Met Asn Leu Ala Gly Gly Al 3 1220 1225 1230 Glu Gly Gly His Asp Leu Thr Ala Ile Cys Asp 1235 1240 1245 Ala Ser Glu Ala Ala Leu Leu Gly Asn Arg Wall Asp For 1250 1255 1260 Leu Ser Glu Gin Lys Pro Asn Leu Asn Ala ile Arg 1265 1270 1275 1280 Ser Leu Glu Ala Val Ile Arg Val His Ser Lys Cys Gly Asp Gly Thr 1285 1290 1295 Leu Glu Leu Arg Leu Lys Asp Leu Gly Gly Thr Leu For His Arg 1300 1305 1310 Gly Gin Phle Arg Cys Gin Pro Gly Asp Leu Leu Leu Wall 1315 -320 1325 Ser Lys Ile Pro Val Ser Asp Pro Gly Ser Asn Gly Glu His For

1330 1335 1340

Pro Val Gly Tyr Pro Leu Ser Pro Pro Gly Ala Ser Arg Ala 1345 1350

• * · · • «· · • · · · · · « • • • · • «· • • • · • * • · • · • · • · • · • · • · • · • · • · Tyr Gin Thr Ala 1365 For Gin Gly Lys Tyr Trp 1370 Gly Cys Met Gin 1375 Arg Leu Ala Ser Cys 1380 For Asp Ser Trp Val Pro Arg 1385 Wall For 1 Gly 390 Ala Asp Lys Glu Glu Val 1395 Glu Ala Val Thr Ala Leu Ala * 5 U G Ser Leu 405 Ser Wall Gly Ile Leu Glu 410 Asp Arg 1_ Pro Glu Gin Leu Val 415 1420 Glu Glu Glu Glu Prc 1425 Met. Asn Leu (2) Information c SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(ii) (A) LENGTH: 1200 residues (B) TYPE: amino acid MOLECULAR TYPE: protein (vi) ORIGINAL SOURCE: (xi) (A) ORGANISM: Homo sapiens Sequence Description: SEQ ID NO: 8:

For ± Gin For Gin For 5 Met Asp Leu Arg Wall 10 Gly Gin Arg For For 15 Dec Wall Glu For For For Glu For Thr Leu Leu Ala Leu Gin Arg For Gin Arg 20 May 25 30 Leu His His His Leu Phe Leu Ala Gly Leu Gin Gin Gin Arg Ser Wall 35 4 0 45 Glu For Met Arg Wall Lys Met Glu Leu For Ala Cys Gly Ala Thr Leu 50 55 60 Ser Lsu Wall For Ser Leu For Ala Phe Ser Ile For Arg His Gin Ser 65 70 75 80 Gin Ser Ser Thr For Cys For Phe Leu Gly Cys Arg For Cys For Gin 85 90 95 Leu Ser Met Asp Thr For Met For Glu Leu Gin Wall Gly For Gin Glu 100 ALIGN! 105 110 Gin Glu Leu Arg Gin Leu Leu His Lys Asp Lys Ser Lys Arg Ser Lys 115 120 125 Glu Wall Ala Thr For Ala Gin For Ser For Thr Ser Gin Wall For Ala 130 135 140

107 «·· ·

• • • · • · · · · • · • · · · · · · • • « • · • · Ala Ala Cys Wall Ala Cys Ala Wall Ala Ser Ser Wall Wall Lys Gin Lys 145 150 155 160 Leu Ala Glu Wall Ile Leu Lys Lys Gin Gin Ala Ala Leu Glu Arg Thr 165 170 175 Wall His For Asn Ser For Gly Ile For Tyr Arg Ser Gin Gly For Cys 180 185 190 Ser Gly Gin Cys For Cys Ser Wall For Thr For Leu Lys Gin For Trp 195 200 205 His Ser Phe Cys Arg Thr Leu Glu For Leu Glu Thr Glu Gly Ala Thr 210 215 220 Arg Ser Met Leu Ser Ser Phe Leu For For Wall For Ser Leu For Ser 225 230 235 240 Asp For For Glu His Phe For Leu Arg Lys Thr Wall Ser Glu For Asn 245 250 255 Leu Lys Leu Arg Tyr Lys For Lys Lys Ser Leu Glu Arg Arg Lys Asn 260 265 270 For Leu Leu Arg Lys Glu Ser Ala For For Ser Leu Arg Arg Arg For 275 280 285 Ala Glu Thr Leu Gly Asp Ser Ser For Ser Ser Ser Ser Thr For Ala 290 295 300 Ser Gly Cys Ser Ser For Asn Asp Ser Glu His Gly For Asn For Ile 305 310 315 320 Leu Gly Ser Glu Ala Leu Leu Gly Gin Arg Leu Arg Leu Gin Glu Thr 325 330 335 Ser Wall Ala For Phe Ala Leu For Thr Wall Ser Leu Leu For Ala Ile 340 345 350 Thr Leu Gly Leu For Ala For Ala Arg Ala Asp Ser Asp Arg Arg Thr 355 360 365 His For Thr Leu Gly For Arg Gly For Ile Leu Gly Ser For His Thr 370 375 380 For Leu Phe Leu For His Gly Leu Glu For Glu Ala Gly Gly Thr Leu 385 390 395 400

• ·

1 $ 8 • • • · • • · • · · • • • · • · • • • · · • · • • • · · • · · • · · • • · • · • · For Ser Arg Leu Gin For 2. e Leu Leu Leu Asp For Ser Gly Ser His 405 410 415 Ala For Leu Leu Thr Wall For Gly Leu Gly For Leu For Phe His Phe 420 425 430 Ala Gin Ser Leu Met Thr Thr Glu Arg Leu Ser Gly Ser Gly Leu His 435 440 445 Trp For Leu Ser Arg Thr Arg Ser Glu For Leu For For Ser Ala Thr 450 4 55 460 Ala For For For For Giy For Met Gin For Arg Just Glu Gin Leu Lys 4 65 4'3 475 480 Thr His Wall Gin Wall ^T o Lys Arg Ser Ala Lys For Ser Glu Lys For 485 490 495 Arg Leu Arg Gin Ile For Ser Ala Glu Asp Leu Glu Thr Asp Gly Gly 500 505 510 Gly For Gly Gin Wall Wall Asp Asp Gly Leu Glu His Arg Glu Leu Gly 515 520 525 His Gly Gin For Glu Ala Arg Gly For Ala For Leu Gin Gin His For 530 535 540 Gin Wall Leu Leu Trp Glu Gin Gin Arg Leu Ala Gly Arg Leu For Arg 545 550 555 560 Gly Ser Thr Gly Asp 'Τ'η τ Wall Leu Leu For Leu A.1 <3. Gin Gly Gly His 565 570 575 Arg For Leu Ser Arg Ala Gin Ser Ser For Ala Ala For Ala Ser Leu 580 585 590 Ser Al d For Glu For Ala Ser Gin Ala Arg Wall Leu Ser Ser Ser Glu 595 600 605 Thr For Ala Arg Thr Leu For Phe Thr Thr Gly Leu Ile Tyr Asp Ser 610 615 620 Wall Met Leu Lys His Gin Cys Ser Cys Gly Asp Asn Ser Arg His For 625 630 635 640 Glu His Ala Gly Arg Ile Gin Ser Ile Trp Ser Arg Leu Gin Glu Arg 645 650 655

109 • • • • · · • · • • · • · · · • • • • · • • · • • • · · • • · • • • · · • · • • « • · • · • * • · • · • · Gly Leu Arg Ser Gin Cys Glu Cys Leu Arg Gly Arg Lys Ala Ser Leu 660 665 670 Glu Glu Leu Gin Ser Wall His Ser Glu Arg His Wall Leu Leu Tyr Gly 675 680 685 Thr Asn Pno Leu Ser Arg Leu Lys Leu Asp Asn Gly Lys Leu Ala Gly 690 695 700 Leu Leu Ala Gin Arg Met Phe Wall Met Leu For Cys Gly Gly Wall Gly 705 710 715 720 For Leu Ala Thr Leu Ser Ala Phe Leu Ala Ser Leu Ala For Thr Wall 725 730 735 For Gin Gly Leu Ser Arg Wall Ser Trp Gly Leu Lys For For For Gly 740 745 750 For Asn For Lys Ser Arg For Al e For Cys For Trp Gly For Gly Arg 755 760 765 Gly Wall Gly Thr Thr For Leu Gly For Gly Ser Cys Wall Lys For Trp 770 775 780 Met Met Arg Ala Leu Thr Leu Ala For Gin Wall Asp Thr Asp Thr Ile 785 790 795 800 Trp A.sn Glu Leu His Ser Ser Asn Ala Ala Arg Trp Ala Ala Gly Ser 805 810 815 Wall Thr Asp Leu Ala Phe Lys Wall Ala Ser Arg Glu Leu Lys Asn Gly 820 825 830 Phe Ala Wall Wall Arg For For Gly His His Ala Asp His Ser Thr Ala 835 840 845 Met Gly Phe Cys Phe Phe Asn Ser Wall Ala Ile Ala Cys Arg Gin Leu 850 855 860 Gin Gin Gin Ser Lys Ala Ser Lys Ile Leu Ile Wall Asp Trp Asp Wall 865 870 875 880 His His Gly Asn Gly Thr Gin Gin Thr Phe Tyr Gin Asp For Ser Wall

885 890 895

Leu Tyr Ile Ser Gly Asn Phe Phe Pro Gly 900 905 910

1 10. · • • • • • * • • • · • · • • • • · · • «· • • • · · • • · • • • · · • · # • • • « • · • · • · • · • · Ser Gly Val Val Gly Val Gly A.la Gly Ser Gly Glu Gly Phe Asn 915 920 925 Val Asn Val Ala Trly Ala Gly Gly Leu Asp Breast For Met Gly Asp For 930 935 94 0 Glu Tyr Leu, Ala Ala Phe Arg Ile Val Wall Mrx. - For Ile Aia Arg Glu 945 950 955 960 Phe Ser Pro Leu Val Leu Val Ser 7 \ ΛΧ S G L y Phe Asp A.la Ala Glu 965 970 975 Gly His Pro Gla Gly Tyr His χ 7 - * Ser A. ± 3 Lys Cys Phe 980 985 990 Gly Tyr Met Thr Leu Ala Gly Gly Ala Wall Wall 995 1000 1005 Leu Ala Glu Gly Gly His Asp Leu Thr Ala Ile Cys Asp Ala Ser 1010 1015 1020 Glu .Ala Cys Val Ala Ala Leu Leu Gly Asn A.r s Wall Asp For Leu Ser 1025 1030 ί_ 035 1040 Glu Glu Gly Lys For Asn Leu A.sn Ala Ile Arg Ser Leu 1045 1050 1055 Glu Ala Val Ile Cys C-iy Asp Gly Thr Leu Ala 1060 1065 1070 Glu Leu Lys Asp Leu Gly Gly Thr Leu For His Arg Gly Gin 1075 1080 1085 Ile Leu Gly Asp Leu Leu Leu Wall Trp Ser 1090 1095 1100 Lys Ile For Val Ser Asp For Gly Ser Asn c-iy Glu His For For Wall 1105 1110 1 115 1120 Arg Gly Tyr Pro Le Pro Ser Pro Pro Asp Gly A._a Ser Arg Ala Tyr Gin 1125 1130 1135 Thr Val Ala Gly Lys Tyr Trp Gly Cys Met Gin Arg Leu Ala 1140 1145 1150

110

Ser Cys Pro Asp Ser Trp Val Pro Arg Val Gly Ala Asp Lys Glu 1155 1160 1165 iíli: ·

Glu Val · Glu Ala Val Thr Ala Lea Ala Ser Lee Gly Ile Leu 117G 1175 1180

Ala Glu Asp Arg Pro Glu Gin Glu Glu Glu Glu Glu Pro Met 1185 1190 1195 1200 ··· ··· · · ·

IV (2) Information about SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(ii) (A) LENGTH: 1,041 residues (B) TYPE: amino acid MOLECULAR TYPE: protein (vi) ORIGINAL SOURCE: (xi) (A) ORGANISM: Homo sapiens Sequence Description: SEQ ID NO: 9:

For 1 Ser A.la Wall For 5 Met Asp Leu Arg Leu 10 A.sp His Gin Phe Ser 15 Dec Leu For Wall Ala Glu For Ala Leu Arg Glu Gin Gin Leu Gin Gin Glu Leu 20 May 25 30 Leu Ala Leu Lys Gin Lys Gin Gin Ile Gin Arg Gin Ile Leu Ile Ala 35 40 45 Glu Phe Gin Arg Gin His Glu Gin Leu Ser Arg Gin His Glu Ala Gin 50 5 5 60 Leu His Glu His Ile Lys Gin Gin Gin Glu Met Leu Ala Met Lys His 65 70 75 80 Gin Gin Glu Leu Leu Glu His Gin Arg Lys Leu Glu Arg His Arg Gin 85 90 95 Glu Gin Glu Leu Glu Lys Gin His Arg Glu Gin Lys Leu Gin Gin Leu 100 ALIGN! 105 110 Lys Asn Lys Glu Lys Gly Lys Glu Ser Ala Vdl. Ala Ser Thr Glu Wall 115 120 125 Lys Met Lys Leu Gin Glu Phe Wall Leu Asn Lys Lys Lys Ala Leu Ala 130 135 140 His Arg Asn Leu Asn His Cys Ile Ser Ser Asp For Arg Tyr Trp Tyr

145 150 155 160

Gly Lys Thr Gin Ser Ser Leu Asp Gin Ser Ser Pro Pro Gin Ser

113 «« · «·················· · ··· ·· ··

165 170 175

Gly Val Ser Thr Ser Tyr Asn His Pro Val Leu Gly Met Tyr Asp Ala 130 185 190

Lys Asp Asp Phe Pro Leu Arg Lys Thr Ala Ser Glu Pro Asn Leu Lys 195 200 205

Argu Ser Ser Argu Ser Lys Gin Lys Val Ala Glu Arg Arg Ser Pro 210 215 220

Leu Arg Arg Lys Asp Gly Pro Val Val Thr Ala Leu Arg Lys Arg

(+420) 225 230 235 240

Leu Asp Val Thr Asp Ser Ala Cys Ser Ser Ala Pro Gly Ser Gly

245 250 255

Ser Ser Pro Ser Ser Gly Ser Ser Gly Ser Ser Gly Asn Gly 260 265 270

Ile Ala Pro Ala Val Pro Ser Ile Pro Ala Glu Thr Ser Leu Ala His 275 280 285

Arg Leu Val Ala Glu Gly Ser Ala Ala Pro Leu Pro Leu Tyr Thr 290 295 300

Asn Ile Thr Leu Gly Leu Pro

305 310 315 320

Ser Ala Gly Thr

(+420) 325 330 335

Ala Leu Gin Gin Arg Leu Phe Pro Gly Thr His Leu Thr Pro 340 345 350

Tyr Leu Ser Thr Ser Pro Glu Glu Arg Asp Gly Gly Ala Ala His Ser 355 360 365

Pro Leu Leu Gin His Met Val Leu Leu Glu Pro Pro Ala Gin Ala 370 375 380

Pro Leu Gly Leu Gly Ala Leu Pro Leu His Ala Gin Ser Leu

385 390 395 400

Val Gly Ala Asp Arg Ser Ser Ser Ile His Lys Leu Arg Gin His

405 410 415

Arg Leu Gly Arg Thr Gin Ser Ala Leu Gin Asn Ala Gin

Ala

Lys

Ile

465

Glu

Tyr

Wall

For

Leu

545

Gin

Ser

Ser

Phe

Thr

625

Ser

Cys

Leu

His

450

For

Thr

Leu

Gin

For

530

Leu

Leu

Phe

Ala

Thr

610

Cys

Ile

Ile

Gin

435

Lys

Lys

Glu

Asp

Wall

515

Arg

Phe

Arg

Gly

Thr

595

Thr

Gly

Trp

Arg

420

His Leu

Gin Gin

Pro Ser

Glu Glu 485

Arg Leu 500

Lys Gin

Glu Val

Arg Gin

Asn Tyr

Gly His 580

Phe Pro

Gly Leu

Ser Ser

Ser Arg 645

Gly Arg 660

425

Val Ile Gin Gin 440

Phe Gin Gin Gin

Glu Pro Ada Arg 470

Leu Arg Glu His

For G.ly Gin Lys 505

Glu Pro Ile Glu 520

Glu Pro Gly Gin

Gin Ala Leu Leu 550

Gin Ala Ser Met

Arg Pro Leu Ser 586

Val Ser Val Gin 600

Val Tyr Asp Thr 615

Ser Ser His Pro 630

Glu Glu Thr

Lys Ala Thr Leu 666

Gin His Gin

Gin Leu Gin 460

Gin Pro Glu 475

Gin Ala Leu 490

Glu Ala His

Ser Asp Glu

Arg Gin Pro 540

Leu Glu Gin

Glu Ala Ala

Arg Ala Gin

Glu Pro Pro

Leu Met Leu 620

Glu His Ala

Gly Leu Arg 650

Glu Glu Leu e e e e e e e

430

Gin Phe Leu Glu 445

Met Asn Lys Ile

Ser His Pro Glu 480

Leu Asp Glu Pro 494

Ala Gin Ala Gly 510

Glu Glu Ala Glu

Ser Glu Gin Glu

Gin Arg Ile His 560

Gly Ile Pro Val 576

Ser Ser Pro Ala 590

Thr Lys Pro Arg 604

Lys His Gin Cys

Gly Arg Ile Gin 63 0

Gly Lys Cys Glu 655

Gin Thr Val His 670

Ser Glu Ala His Thr Leu Leu Thr Gly Thr Asn For Leu Asn Arg Gin

115

4 4 4 »4 4 4 4

675 680

Lys Leu Asp Ser Lys Leu Leu 690 695

Arg Leu Pro Cys Gly Gly Val Gly 705,710

Glu Val His Ser Ala

Glu Leu Val Lys Val Ala Thr 740

Val Val Pro Pro Gly His His 755,760

Phe Cys Tyr Phe Asn Ser Val Ala 770 775

Arg Leu Ser Val Lys Ile Leu 785,790

Gly Asn Gly Gin Gin Ala Phe 805

Met Ser Leu His Arg

Ala Pro Asp Glu Val Gly Thr Gly 835 840

Met Ala Phe Thr Gly Gly Leu Asp 850 855

Leu Ala Ala Thr Val Val 865 870

For Asp Val Val Leu Val Ser Ser 835

Pro Thr Pro Leu Gly Gly Tyr Asn 900

Leu Thr Lys Gin Leu Met Gly Leu 915 920

44 44 44 44 44 44 44 44

5

Gly Ser Leu Ala

Val Asp Ser Asp Thr Ile Trp Asn 715 720

Arg Leu Ala Val Gly Val Val 730 735

Gly Glu Leu Lys Asn Gly Phe Ala 745,750

Ala Glu Glu Ser Thr Pro Met Gly 7

Val Ala Ala Lys Leu Leu Gin Gin 780

Ile Val Asp His Asp 795 800

Tyr Ser Asp Pro Ser Val Leu Tyr 810 815

Gly Asn Phe Pro Gly Ser Gly 825 830

For Gly Val Gly Phe Asn Val Asn 845

Pro Pro Gly Asp Ala Glu Tyr 860

Met Pro Ile Ala Ser Glu Phe Ala 875,880

Gly Phe Asp Glu Gly His 890 895

Leu Ser Ala Arg Cys Phe Gly Tyr 905 910

Ala Gly Gly Ile Val Leu Ala 925

Leu Glu Gly Gly His Asp Leu Thr Ala

116 · · · 4 4 4 4 4 4 4 4 4 4 9 9 9 9 9 9 4 4 4 4 4

930

Cys Val 945

Val Leu

Val Met

Ser Thr

Glu Ala 1010

For Al 1025

Leu

935

940

Ser Ala Leu Leu 950 Gly Asn Glu Leu Asp 955 For Leu For Glu Lys 960 Gin Gin Arg For Asn Ala Asn Ala Wall Arg Ser Met du Lys 965 970 975 Glu Ile His Ser Lys Tyr Trp Arg Cys Leu Gin Arg Thr Thr 980 985 990 Ala Gly Arg Ser Leu Ile Glu Ala Gin Thr Cys Glu Asn Glu 995 1000 1005 Glu Thr Wall Thr Al a Me- Ala Ser Leu Ser Wall Gly Wall Lys 1015 1 , 020 Glu Lys Arg For Asp du du For Met du Glu Glu For For 1030 1035 1040

··· · · · · ·

Claims (14)

PATENT CLAIMS • · 117 A caspase-8 interacting protein or isoform, allelic variant, fragment, functional analog, mutant or derivative thereof that is capable of interacting with caspase-8 subunit 1 and / or 2. The protein of claim 1 which is human Nacetylglucosamine-6-phosphate deacetylase or an isoform thereof, allele variant, fragment, functional analog, mutant or derivative thereof. The protein of claim 1, which comprises the amino acid sequence shown in Figure 2, 3, 5B or 6. The protein of claim 1 which is encoded by clone p74 or a splice-modified variant thereof The protein of claim 4, wherein the splice variant is selected from p16 and p43 clones. The protein of claim 1 encoded by clone p27, p70 or p79. The protein of claim 1, which comprises the amino acid sequence of the Tip-60 protein excluding amino acids 94-145. The protein of claim 1, which encodes clone L7, L12, M26, B4, B17, J40, B13, B37, or B33. The protein of claim 1, which is cleaved in vitro by caspase 8. The protein of claim 1, which is cleaved in vivo by caspase-8. The isolated DNA sequence encoding the protein of claim 1. An isolated DNA sequence encoding a protein according to any one of claims 2 to 10. An isolated DNA sequence comprising the DNA sequence shown in Figure 2. An isolated DNA sequence comprising the DNA sequence shown in Figure 3. • · 118 An isolated DNA sequence comprising the DNA sequence shown in Figure 5A. An isolated DNA sequence capable of hybridizing to a DNA sequence according to any one of claims 11 to 14 under moderately stringent conditions. A vector comprising the DNA sequence of any one of claims 11 to 16. A eukaryotic or prokaryotic host cell comprising the vector of claim 17. A method for producing a caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative of claim 1, comprising culturing the host cell of claim 18 under conditions that allow the production of said protein, affecting posttranslational adjusting, if necessary, to obtain said protein, isoform, allele variant, fragment, functional analog, mutant or derivative, isolating said protein, isoform, allele variant, fragment, functional analog, mutant or derivative. The method of claim 19, wherein the cell is a prokaryotic cell. The method of claim 19, wherein the cell is a eukaryotic cell. The method of claim 21, wherein the cell is a mammalian, insect, or yeast cell. The method of claim 22, wherein the cell is a HeLa or 293 T HEK cell. The method of claim 19 wherein the promoter is a CMV promoter. A caspase-8 interacting peptide comprising at least 4 consecutive amino acids of the protein of claim 1. The peptide derivative of claim 23. • · A peptide derivative according to claim 26 which is capable of forming a covalent bond with caspase-8 upon contact with the caspase-8. A nucleozyme specific for a nucleotide sequence corresponding to the DNA sequence of any one of claims 11 to 16. A nonsense oligonucleotide comprising at least 9 nucleotides of a sequence corresponding to the DNA sequence of any one of claims 11 to 16. An antibody directed to an epitope of a protein according to claim 1. 31. An immunoassay suitable for detecting a caspase-8 interacting protein comprising the antibody of claim 30 as an agent. 32. Immunological test suitable for detection of caspase-8, mark ic i se by comprising a peptide according to any of claims 25 to 27. 33. Immunological test suitable for detection of caspase-8, mark ic i se by comprising a protein according to any of of claims 1 to 10. 34. Method of identification of proteins Caspase-8 interacting mark ic i s e t i m, that includes and) yeast preparation cells, which contains a reporter gene linked to a promoter that contains a DNA sequence motif, b) expressing the p20 subunit of said caspase-8 in said yeast cell, c) expressing a fusion protein of the DNA binding region and the p10 and / or p20 subunit of said caspase-8 in said yeast cell, wherein said DNA binding region is capable of binding to said DNA sequence motif, d) expressing, as appropriate, the unfused p10 or p20 subunit of said caspase-8, e) transforming the culture of said yeast cell with a library comprising an expression vector directing the expression of a fusion protein comprising a cDNA library and transcription, testing the culture of the transformed yeast cell activator where the reporter gene is activated, g) isolating the yeast cell described in f) and further isolating the caspase-8 interacting protein expressed in its predator vector. The caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative of claim 1 or the ribozyme of claim 28 or the nonsense oligonucleotide of claim 29 or the antibody of claim 30 suitable for use in modulating caspase8 activity. The caspase-8 interacting protein, isoform, allelic variant, fragment, functional analog, mutant or derivative of claim 1 or the ribozyme of claim 28 or the nonsense oligonucleotide of claim 29 or the antibody of claim 30 suitable for use in modulating TNF receptor activity or effect mediated by Fas. The caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative of claim 1 or the ribozyme of claim 28 or the nonsense oligonucleotide of claim 29 or the antibody of claim 30 suitable for use in the treatment of apoptosis. A caspase-8 interacting protein, isoform, allelic variant, fragment, functional analog, mutant or derivative of claim 1 or a ribozyme of claim 28 or a nonsense oligonucleotide of claim 29 or an antibody of claim 30 suitable for use as a medicament. A caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative according to claim 1 or a ribozyme according to claim 28 or a nonsense oligonucleotide according to claim 29 or an antibody according to claim 30 suitable for use as a medicinal product in 121 treatment of multiple sclerosis with primary oligodendrogliopathy, autoimmune uveoretinitis, diabetes, psoriasis, autoimmune myocarditis I, HCV-mediated chronic hepatitis, chronic castritis such as gastritis type A, mixed connective tissue disease (MCTD), Crohn's disease, or ulcerative colitis. Use of a caspase-8 interacting protein, isoform, allele variant, fragment, functional analog, mutant or derivative according to claim 1 for the isolation, identification and cloning of another protein of the same class.