JPH10500568A - TNF / NGF superfamily receptor and soluble oligomer TNF / NGF superfamily receptor modulator - Google Patents

Abstract

  (57) [Summary] The present invention relates generally to novel proteins capable of binding to the p55 and p75 TNF receptors and the intracellular domain of the Fas receptor and modulating the functions of the p55 and p75 TNF receptors and the Fas receptor, and encoding the proteins. DNA sequence to be used. The present invention also relates to novel soluble oligo-TNF receptors, oligo-Fas receptors and oligo-receptors having a mixture of TNF and Fas receptors. The invention furthermore relates to the preparation and use of all the protein and DNA sequences mentioned above.

Description

DETAILED DESCRIPTION OF THE INVENTION   TNF / NGF superfamily receptors and   Soluble oligomer TNF / NGF superfamily   Receptor modulators                                 Technical field   The present invention generally relates to receptors belonging to the TNF / NGF superfamily of receptors. And the art of controlling the biological function of these receptors. TNF / NGF Superfamily receptors include, for example, tumor necrosis factor of p55 and p75 Receptor (TNF receptor) and FAS ligand receptor (FAS / APO1 or There is a receptor such as FAS receptor (hereinafter referred to as FAS receptor). Sa More specifically, the present invention relates to p55TNF receptor and p75TNF receptor and And Fas receptor intracellular domains (ICs) p55IC, p75IC and FasIC) and p55T Modulates the function of NF and p75TNF and Fas receptors New proteins that can be used. Complete p55-TNF receptor p One of the proteins that can bind 55IC is a p55IC molecule or, for example, p5 The shape of its part, such as the so-called "death domain" (DD) of the 5IC P55IC itself. Therefore, the present invention relates to the intracellular expression of the p55TNF receptor. Independent of ligand (TNF) by domain (p55IC) or part thereof Can be induced into cells in a new way Regarding regular TNF-related effects. The present invention also relates to these novel p55TNF receptors. And p75TNF receptor binding protein and Fas receptor binding protein (In the present specification, p55IC binding protein and p75IC binding protein are referred to respectively. And FasIC binding proteins).   In another aspect, the present invention also provides novel soluble oligomeric TNF receptors and oligosaccharides. Or containing a mer FAS receptor and a mixture of a TNF receptor and a FAS receptor. The present invention relates to a sesame receptor, a use thereof, and a production method thereof.                        Background of the Invention and Prior Art   Tumor necrosis factor (TNF-α) and lymphotoxin (TNF-β) (hereinafter TNF-β) Means both TNF-α and TNF-β) has many effects on cells Multifunctional pro-inflammatory cytokines (pro-inflammats) mainly produced by mononuclear phagocytes ory cytokine) (Ion Gresser edited by "Interferon (interferon) 7 ”, pp. 83-122 (Academic Press) Wallach D. (1986), Inc., London); And Beutler and Cerami (1987)). With TNF-α Both TNF-βs act by binding to specific cell surface receptors. To start. Some of these effects are considered beneficial to the organism. That is, for example, tumor cells or cells infected with a virus by these actions Are destroyed and the antimicrobial activity of granulocytes is increased. In this way, TNF feels like a tumor Dye factor It is involved in the defense of organisms against and recovery from damage. Therefore TNF binds to the receptor for TNF on the surface of tumor cells and kills tumor cells. It can be used as an antitumor agent in applications that trigger events leading up to it. TNF is It can also be used as an anti-infective agent.   However, both TNF-α and TNF-β also have deleterious effects. TNF-α exceeded When produced in excess, it is clear that it can act as a serious pathogen in some diseases. is there. In other words, the action of TNF-α mainly on the vascular system is It is known to be a major cause of symptoms (Tracey et al. (1986)). . In some diseases, TNF regulates adipocyte activity and causes anorexia TNF-α may be cachetin (cache), since excessive weight may be lost (cachexia). tin). In addition, TNF-α has been shown to damage tissues in rheumatic diseases. As wound mediators (Butler and Cerami; (1987)) and As a major mediator of damage seen in graft-versus-host reactions (Pique t) et al. (1987)). In addition, TNF is involved in inflammatory processes and And are known to be involved in many other diseases.   P55TNF receptor and p75T, two differently expressed receptors The NF receptor specifically captures both TNF-α and TNF-β, Initiates and / or mediates the biological effect. These two receptors have different structures. Have different intracellular domains, and these receptors have different signaling (See reports below, Hohman) (1989); Angelman et al. (1990). Bruckhaus et al. (1990), Leotscher et al. (1990) ), Schall et al. (1990), Nofar et al. (1990) (Smith et al. (1990) and Heller et al. (1990)). However, its cellular mechanisms, such as p55TNF receptor and p75TNF receptor Various proteins involved in the intracellular signaling of the body and possible involvement Certain other factors have yet to be elucidated (as described below, first, p75 New proteins that can bind to IC and p55IC are described). This cell Intracellular signaling usually involves the ligand, ie, TNF (α or β) Occurs after binding to the body and finally leads to a response of cells to TNF It is involved in initiating a cascade of reactions.   The cytocidal effect of TNF has been studied so far. In most cells, this effect is mainly triggered by the p55TNF receptor. It is. To the extracellular domain (ligand binding domain) of p55TNF receptor The antibodies themselves induce a cell killing effect (see EP 412486), which Is correlated with the efficiency of receptor cross-linking by This is considered to be the first step in the generation of the process of gnarling. Further mutation (Brakebusch et al. (1992); Tartagli) (Tartaglia et al. (1993)), the biological function of the p55TNF receptor Intracellular domain integrity (integrity), TNF has a cell killing effect Intracellular signaling is initiated by two or more intracellular domains of the p55TNF receptor. It was suggested that this would occur as a result of the meeting of the members. In addition, TNF (α and β) Be formed as a homotrimer, so its ability to bind and crosslink receptor molecules Thus, inducing intracellular signaling via the p55TNF receptor, This suggests that receptor aggregation occurs. What are p55IC and p55DD TNF-related effects (TNF-assoc) in cells in a ligand-independent manner Whether or not an iated effect is induced will be described below.   Another member of the TNF / NGF superfamily of receptors is Fas FAS receptor (FAS receptor), which is also called antigen, and is generated in various tissues. Homologous to several cell surface receptors expressed and including TNF and NGF receptors It is. FAS receptors mediate cell death in the form of apoptosis (Itoh) (1991)) and the negative selector of autoreactive T cells during T cell maturation. -It seems to work as a (Selector). FAS receptor is a T cell that recognizes self antigen Mediates apoptotic death. FAS receptor gene (Ipr) Is mutated Then, mice are given systemic lupus erythematosus (SLE), a human autoimmune disease Have been found to cause lymphoproliferative disorders similar to (Watanabe-Fukunaga) (Watanabe-Fukunaga) et al. (1992)). The ligand for the FAS receptor is In particular, killer T cells (ie, cytotoxic T lymphocytes: CTL) Cell-surface associated molecule As such, when such CTLs come into contact with cells bearing FAS receptors, It can induce apoptotic death of FAS receptor-bearing cells. Furthermore, FA Monoclonal antibodies specific to the S receptor have been prepared and Mouse was transformed with a cDNA encoding the human FAS receptor. Inducing apoptotic cell death in cells bearing FAS receptors, including certain cells (Itoh et al. (1991)).   Various normal cells other than T lymphocytes express FAS receptors on their surfaces. Has been found to be killed by the induction of this receptor. Such death Inducing uncontrolled processes can result in tissue damage in certain diseases, such as acute It is thought to cause hepatocyte destruction in hepatitis. Therefore, FAS acceptance Finding ways to reduce the body's cytotoxic activity could be used therapeutically.   Conversely, certain malignant cells and HIV-infected cells carry FAS receptors on their surface. Antibody to the FAS receptor, ie the FAS receptor Ligands are required to induce FAS receptor-mediated cytotoxic effects on these cells. Can be used to provide a means to combat such malignant or HIV-infected cells. (Itoh et al. (1991)). Therefore, FAS receptor cytotoxicity Finding other ways to increase activity could be used for treatment.   For example, TNF (α or β) and FAS receptor ligand in the above pathological condition It has long been thought necessary to provide a way to regulate the cellular response to Have been However, when TNF or FAS receptor ligand is overexpressed, TNF Alternatively, it is desirable to inhibit the cell killing effect induced by the FAS receptor ligand On the other hand, it is desirable to enhance TNF action in other conditions, such as healing a wound. Or, in the case of FAS receptors on tumor cells or HIV infected cells, FA It is desirable to enhance S receptor-mediated action.   We have made a number of methods (eg European Patent Application, E P186833, EP308378, EP398327 and EP412486 Inhibiting TNF binding to its receptor using anti-TNF antibodies Or by the soluble TNF receptor (especially the soluble extracellular domain of the receptor) Competes with TNF binding to cell surface bound TNF receptor using Have regulated the detrimental effects of TNF. In addition, TNF is a cell That TNF needs to bind to its receptor to elicit an effect Based on this, we modulate the activity of the TNF receptor, A method for regulating the action of NF (for example, see EPO 568925) has been produced. Was. Briefly, EPO 568925 is a signal at the TNF receptor Regulates the transmission and / or blockage of proteins to allow peptides or other molecules to Interacts with the receptor itself or effector proteins that interact with the receptor To modulate the normal function of the TNF receptor. EP O568925 contains the extracellular domain of the p55TNF receptor, transmembrane. In the run and intracellular domains Construction and characterization of various mutants of the p55TNF receptor having a mutated portion have been described. ing. Thus, a region within the domain of the p55TNF receptor is where the receptor is Function, ie, capture ligand (TNF), and then signal is transmitted The signaling takes place to ultimately result in the observed TNF effects on the cells Confirmed to be essential. In addition, various regions within the domain of the TNF receptor Capable of binding and involved in controlling or regulating the activity of the TNF receptor Several methods for isolating and identifying proteins, peptides or other factors are also described. You. Isolate and clone DNA sequences encoding such proteins and peptides The expression vector used to produce these proteins and peptides. A method of constructing; and binding to the TNF receptor or various regions of the TNF receptor. Methods for producing antibodies or fragments thereof that interact with proteins and peptides Some are also described in EPO 568925. But EPO 568925 Binds to the intracellular domain of the TNF receptor (eg, the p55TNF receptor) Binds to actual proteins and peptides and the intracellular domain of the TNF receptor Yeast two-hybrid for isolating and identifying such proteins or peptides No mention is made of the yeast two-hybrid approach. Similarly, Later, proteins or peptides that can bind to the intracellular domain of the FAS receptor Nothing is disclosed.   Therefore, if it is desired to suppress the action of TNF or FAS receptor ligand, Reducing the amount or activity of TNF or FAS receptors on the cell surface Is desirable However, if it is desired to enhance the action of TNF or FAS receptor ligand, TNF It is desirable to increase the amount or activity of the receptor or FAS receptor. This purpose For this reason, we have recently discovered that both the p55TNF receptor and the p75TNF receptor Determine the sequence of the promoter and analyze it to determine whether it is specific for various transcription factors. The main sequence motif was discovered. The expression of these TNF receptors is therefore It can be controlled at the promoter level. That is, transcribed by the promoter To reduce the number of receptors, Can be increased (IL104355 and IL109633 and this (See corresponding unpublished EP and PCT applications). FAS receptor gene Corresponding research results on the regulation of FAS receptors at the motor level Must be reported.   In addition, the following must be stated. That is, tumor necrosis factor (T NF) The FAS receptor and its structurally similar receptors are The destructive activator (destructive a is known to be triggered in cells when stimulated by ctivities) However, little is known about the mechanism of this triggering. Mutation research results The result is that the FAS receptor and the p55TNF receptor (p55 receptor) Showing that gnarling occurs in distinct regions within their intracellular domain (Brakebusch et al. (1992); Tartaglia (1993); Itoh and Nagata (1993)). This There is sequence similarity in these regions ("dead domains"). FAS receptor and p55 The "dead domains" of both of the receptors tend to self-associate. This self-assembly is necessary to start signaling (Song et al.) (1994); Wallach et al. (1994); Boldin et al. (1995) as well as below) and high levels of receptor expression Can trigger ligand-independent signaling (Boldin) (Described below (1995)) It is clear that aggregation of the receptor is promoted. is there.   Therefore, prior to the present invention, it belonged to the TNF / NGF superfamily. TNF or the action of ligands, eg, by mediating intracellular signaling processes Proteins that can regulate the action of FAS receptor ligands on cells are Not provided. Note that the signaling is probably mostly for TNF / NGF The intracellular domain (IC) of a receptor belonging to the superfamily of receptors, for example Intracellular domains of TNF receptor, p55TNF receptor and p75TNF receptor Intracellular domains (p55IC and P75IC, respectively) and FAS-IC So it is dominated.   Accordingly, one object of the present invention is to provide an intracellular domain of TNF and FAS receptors. Binding to TNF and binding of TNF to its receptor or FAS ligand Is involved in the intracellular signaling process initiated by binding to its receptor. To provide proteins that are currently believed to be conferring.   Another object of the present invention is to provide these intracellular domain binding proteins (IC binding proteins). Is a positive signal effector (ie, triggers signaling) If necessary, to suppress the signaling process when desired, or If the protein is a negative signal effector (ie, suppresses signaling) In some cases, this can be used to facilitate the signaling process when desired. Antagonists (eg, antibodies) to these intracellular domain binding proteins To provide.   Yet another object of the present invention is to involve further downstream in said signaling process. To isolate and characterize other proteins or factors that may be The use of these IC binding proteins and / or the signaling process Further upstream, these IC binding proteins bind to other receptors (eg, other T-binding proteins). NF receptors or analogous receptors) (and thus the function of these receptors These IC binding proteins can be used to isolate and identify It is that you are.   Further, an object of the present invention is to provide a polyclonal antibody against the IC binding protein. IC binding as an antigen for preparing human and / or monoclonal antibodies The use of protein. This antibody, on the other hand, has different origins, such as cells Purify new IC binding proteins from extracts or transformed cell lines Can be used for   Further, these antibodies can be used for diagnosis. For example, TNF / NGF receptor Abnormal function of cellular actions mediated by receptors in the body superfamily Do Can be used to identify obstacles related to   Still another object of the present invention is to provide a method for treating TNF or FAS ligand-induced symptoms. A medicament comprising the IC binding protein for use in treatment or prevention Compositions and compositions comprising an antagonist of an IC binding protein are provided. These compositions are, for example, IC binding tags contained in the compositions. Depending on the nature of the protein or its antagonist, TNF or FAS To enhance the action of Gand or inhibit the action of TNF or FAS ligand Can be used for   Still another object of the present invention is to provide a soluble oligomer TNF receptor, oligomer F Uses an oligomer that is an AS receptor or a mixture of a TNF receptor and a FAS receptor TNF or FAS receptor produced endogenously or administered exogenously Reveal other ways to remove or antagonize (attenuate) body ligands Is Rukoto. In this regard, one of the attempts in this area is to address the effects of TNF. TNF-binding protein called TBP-I reported to be able to antagonize It has to be mentioned that the isolation of proteins and the production by recombinant methods No. This antagonism measures the decrease in cytotoxic activity of TNF and Measured by measuring the degree to which NF inhibits binding to its receptor. (EP30378). TBP-I is capable of culturing cells at a concentration of several nanograms per ml. Protecting against the toxicity of TNF and using TNF-α and TNF-β simultaneously Both cytokines were reported to inhibit binding to cells. TBP- Further testing of the mechanism by which I works, TBP-I does not interact with target cells, but binds specifically to TNF, Block TNF function by competing with TNF receptor for TNF It became clear.   As a result, it was found that the two active ingredients were present by different purification methods. Was. That is, a second TNF-binding protein called the first TBP-I and the TBP-II Quality (first described in EP 398327). Both of these proteins are T Protects against cell killing of NF in vitro and protects TNF-α against TNF-α It binds more effectively than -β. According to the SDS PAGE analysis results, these 2 Species proteins TBP-I and TBP-II have very similar molecular sizes However, these two proteins have no immunological cross-reactivity, N-terminal Different amino acid sequences and different amino acid compositions Can be distinguished.   However, the previously found soluble TNF binding protein is monomeric, Since it can only bind to one monomer of the natural ligand TNF homotrimer, The two active monomers to which the TNF binding protein is not bound still TNF activity retains TNF activity (ie, incomplete neutralization). ). Furthermore, it is known that the molecule is a homotrimer and is bound to the cell surface. No soluble FAS receptor that can bind to the FAS receptor ligand Not disclosed.   The so-called “dead domain” of p55-IC (Tataglia (1993)) As previously disclosed, according to the present invention, p55-IC and its "dead domain" " Signals that self-associate and this self-assembly triggers cell cytotoxicity (cytotoxis) Was not stated to be primarily involved. In addition, this publication contains Oligomeric TNF receptor or soluble oligomeric FAS receptor or the same The possibility of producing a mixture of oligomers is not mentioned and p55 -Other TNF-related effects elicited by the IC or a part thereof, such as I Induction of L-8 gene expression, that is, the present invention has not been disclosed at all. Similarly, the book Other publications published after the filing date of the invention include the aggregation of p55IC (ie, Association) Performance was disclosed, but as described above, soluble oligomeric TNF receptors or Using p55-IC to produce a Fas receptor, or using p5 Other TNs triggered in a ligand-independent manner by 5-IC or portions thereof It was not related to F-related effects.                               Summary of the Invention   According to the present invention, p55TNF receptor, p75TNF receptor and FAS Novel protein capable of binding to the intracellular domain of each receptor: p55IC binding Fusion protein, p75IC binding protein and FAS-IC binding protein discovered. These p55IC binding proteins, p75IC binding proteins and And FAS-IC binding proteins, wherein TNF is a p55TNF receptor and / or After binding to the p75TNF receptor or when the FAS receptor ligand is Mediates or regulates the intracellular signaling processes that normally occur after binding to By measuring the effect of TNF or FAS receptor ligand on cells, It can act as a heater or modulator. In addition, unexpected Surprisingly, p55IC and FAS-IC can self-associate. And fragments of p55IC and FAS-IC can also bind to p55IC, The so-called killing domain (DD) in the IC of these receptors, ie p55DD And FAS-DD were found to be able to bind to p55-IC. But Thus, p55IC and FAS-IC and their fragments were also used for p55IC and FAS-I. C is a protein that can bind to TNF or FAS receptor ligands. It is a modulator of action on vesicles.   Further, one of the novel proteins of the present invention (55. 11 proteins Binding to the intracellular domain of the p55-TNF receptor. The layers have been fully described (see Example 1).   In yet another aspect, the invention relates to the intracellular domain of the p55TNF receptor (p5 5-IC), the so-called p55-IC “killed” in the region contained in p55-IC Domain "and the intracellular domain of the Fas / APOI receptor (Fas-IC), And the so-called Fas-IC “dead domain” of the region contained in the Fas-IC Is an invention based on the finding that it can self-associate. Therefore Contains at least one extracellular domain of the TNF receptor at one end and the other At least two of said self-associating intracellular domains or portions thereof Soluble oligomer TN which is a fusion product containing The F receptor can be constructed using standard recombinant DNA techniques. The self-association The sex intracellular domain or portion thereof is self-associated and has at least two linked together. An oligomer having two of said fusion products is provided. Therefore such soluble The oligomeric TNF receptor is composed of two models of naturally occurring TNF homotrimers. It can neutralize the activity of TNF by being able to bind to the nomer. TNF activity Neutralizing is associated with endogenous overproduction or exogenously high administration of TNF. Is desirable in all of the above conditions when administered in And Furthermore, the receptor for the soluble oligomer of the present invention effectively enhances TNF. Capturing is when TNF is administered due to its beneficial effects, For example, when treating a tumor, exogenously added TNF is captured and desired. Helps to produce a slow release. Similarly, standard recombinant DNA methods Contain at one end at least two extracellular domains of the FAS receptor And the other end has the self-associating intracellular domain or a portion thereof Construction of oligomeric FAS receptors that are fusion products containing at least two Can also. Note that the self-associating intracellular domain or a part thereof Providing an oligomer having at least two of said fusion products linked together . Thus, such oligomeric FAS receptors are naturally occurring FAS receptors. FAS is capable of binding to two monomers of the homotrimer of the ligand. Effectively neutralizes the activity of receptor ligands. Excessive amount of FAS receptor ligand Unwanted side effects In all of the above conditions, neutralizing the activity of the FAS receptor ligand desirable. In a similar manner, and possible association between TNF and FAS receptor ligands -Induced effects on cells and thus TNF and FAS receptor ligands Recent reports of cell surface binding to cell surface binding to their receptors. In view of the report, a mixture having specificity for both TNF and FAS receptor ligand Oligomeric receptors can be constructed using standard recombinant DNA techniques. Like The mixed oligomer has at one end the extracellular of at least one TNF receptor. A domain and at least one extracellular domain of a FAS receptor, and At least two of said self-associating intracellular domains or portions thereof at the other end Is a mixture of the fusion products. And its self-associating intracellular domain At least two of said fusions are self-assembled and linked together A mixed oligomer containing the product is provided. Therefore, such a mixture Gomer comprises at least one monomer of TNF and at least one FAS receptor Because the ligand monomer can be captured simultaneously, the amount of these two cytokines In the case of such conditions, which cause undesirable cellular effects due to excess, Reduces or effectively neutralizes TNF and FAS receptor ligand activity at the cell surface Can be As mentioned above, FAS receptor ligands usually bind at the cell surface Recent reports also report cell surface-bound TNF. So this These mixed TNF / FAS receptor oligomers bind TNF and FAS receptor on the cell surface. Especially for neutralizing ligand activity Useful for   Therefore, the present invention relates to tumor necrosis factor / nerve growth factor (TNF / NGF) One or more intracellular domains of one or more receptors belonging to the perfamily receptor It provides a DNA sequence encoding a protein capable of binding to the main.   Specifically, the present invention   (a) Code of intracellular domain binding protein of native TNF receptor A cDNA sequence derived from the   (b) hybridization with the DNA of (a) under moderately stringent conditions And binding to the biologically active TNF receptor intracellular domain binding protein DNA sequence to be loaded, and   (c) As a result of the genetic code, degenerate with the DNA sequence defined in (a) and (b) (degenerate rate) and biologically active TNF receptor intracellular domain binding protein DNA sequence to encode And DNA sequences selected from the group consisting of:   The present invention also provides   (a) Coding region of intracellular domain binding protein of natural FAS receptor Derived cDNA sequence,   (b) Hybridization with the cDNA of (a) under moderately stringent conditions And a biologically active FAS receptor intracellular domain binding protein DNA sequence to be loaded, and   (c) as a result of the genetic code, degenerate with the DNA sequence defined in (a) and (b), and Detailed Description of Biologically Active FAS Receptors A DNA sequence encoding an intracellular domain binding protein, And DNA sequences selected from the group consisting of:   In an embodiment of the invention, these DNA sequences are the p55TNF receptor, p7 Encoding 5TNF and FAS receptor intracellular domain binding proteins For example, the proteins named herein: 55. 1, 55. 3, 55. 11 , 75. 3, 75. 16, with DNA sequences encoding F2, F9 and DD11 is there.   The invention also provides a protein encoded by any of the above sequences of the invention. Or an analog or derivative thereof, wherein said protein, analog The body and derivatives may comprise one or more TNF or FAS receptors. Can bind to multiple intracellular domains. Embodiments of this aspect of the invention include those described herein. Proteins named in the book: 55. 1, 55. 3, 55. 11, 75. 3, 7 5. 16, F2, F9 and DD11; analogs and derivatives thereof.   The present invention also relates to the above-mentioned protein of the present invention comprising the DNA sequence of the present invention. Vectors that load in a suitable eukaryotic or prokaryotic host cell. A vector which can be expressed; a transformed eukaryotic cell containing said vector. Or a prokaryotic host cell; and the production of a protein, analog or derivative of the invention. The transformed host cell is grown under currently suitable conditions to obtain the protein. Post-translational modifications of the protein as needed, and then the expressed A protein, analog or derivative from the culture medium of said transformed cells or Proteins of the present invention by extracting from the cell extract of the transformed cells It provides a method for producing an analog or derivative.   In another aspect, the invention is specific for the proteins of the invention, analogs and derivatives thereof. Antibodies or active derivatives or fragments thereof.   In another aspect of the invention, the DNA sequences according to the invention or these sequences are encoded Provides a variety of uses for proteins that There is something.   (i) TNF or FAS reception on cells carrying TNF receptor or FAS receptor A method of modulating the action of a body ligand, comprising a protein, analog and One or more proteins, analogs or derivatives selected from the group consisting of derivatives Treating said cells with the body, and wherein said protein is p55IC, p55IC. 55DD, FAS-IC or FAS-DD, or an analog or derivative thereof And all the proteins are of the TNF receptor or FAS receptor. Regulates TNF or FAS receptor activity by binding to the intracellular domain; Treating said one or more proteins, analogs or derivatives intracellularly. Introduction into said cells in a form suitable for administration, or said one or more tans The DNA sequence encoding the protein, analog or derivative can be transformed into a suitable expression vector A regulatory method comprising introducing into said cells in a state;   (ii) TNF or FAS reception on cells bearing TNF receptor or FAS receptor A method of regulating the action of a body ligand. Treating said cells with the antibody of the present invention or an active derivative or fragment thereof. Adjusting method comprising:   (iii) TNF or FAS reception on cells bearing TNF receptor or FAS receptor A method of modulating the action of a receptor ligand, comprising the steps of: Encoding antisense sequences, or also p55IC, p55DD, FAS-IC Or an oligonucleotide sequence encoding an antisense sequence of the FAS-DD sequence Treating the cells with a column, wherein the oligonucleotide sequence comprises TNF At least one of the receptor or FAS receptor intracellular domain binding protein A regulatory method capable of blocking expression;   (iv) TNF or FAS reception on cells carrying TNF receptor or FAS receptor A method of modulating the action of a body ligand,   (a) encoding a virus surface protein capable of binding to a specific cell surface receptor And an antisense sequence of at least a portion of a sequence of the invention. Oligonucleotide sequences and p55IC, p55DD, FAS-IC or Is an oligonucleotide sequence encoding an antisense sequence of the sequence of FAS-DD Constructing a recombinant animal virus vector carrying a sequence selected from When a nucleotide sequence is introduced into the cell by the virus, TNF receptor Expression of at least one of the body or FAS receptor intracellular domain binding protein Can be shut off;   (b) infecting the cell with the vector of (a); Adjusting method comprising:   (v) TNF or FAS reception on cells carrying TNF receptor or FAS receptor A method of modulating the action of a somatic ligand comprising a protein, analog or derivative of the invention MRNA sequences encoding the body and p55IC, p55DD, FAS-IC Or a sequence selected from mRNA sequences encoding FAS-DD The cells with an appropriate ribozyme-encoding vector having a unique sequence Wherein the ribozyme sequence interacts with the mRNA sequence. And the mRNA sequence can be cleaved, resulting in the protein of the present invention. Expression of proteins, analogs or derivatives or p55IC, p55DD, FAS-I A method of regulating the expression of C or FAS-DD;   (vi) a method of treating tumor cells or HIV infected cells or other pathological cells. What;   (a) is capable of binding to a tumor cell surface receptor or an HIV infected cell surface receptor, Or viral surface proteins capable of binding to other cell surface receptors of other pathological cells Coding sequence, as well as a protein, analog or derivative of the invention. Sequences of the present invention and p55IC, p55DD, FAS-IC, FAS- A sequence encoding DD or a biologically active analog or derivative thereof. Constructing a recombinant animal virus vector carrying the selected sequence, Protein, analogs or derivatives thereof, p55IC, p55DD, FAS-IC, F AS-DD, an analogue or derivative thereof, may be used as the tumor cell or HIV-infected cell. Can be killed when expressed in vesicles or other diseased cells;   (b) replacing the vector of (a) with the tumor cells or HIV-infected cells or other cells; Infect infected cells; A method comprising:   (vii) a protein capable of binding to the intracellular domain binding protein according to the present invention, A method for isolating and identifying a factor or receptor; Bind to the affinity chromatography substrate and bind the bound protein Contacting with the cell extract, and then applying the cell extract to the bound protein. Affinity to elute, isolate and analyze native proteins, factors or receptors A method comprising applying a chromatography method;   (viii) Isolating a protein capable of binding to the intracellular domain binding protein of the present invention A sequence encoding said intracellular domain binding protein To the first hybrid vector, live cDNA or genomic DNA The sequence from the rally is transferred to a second hybrid vector, To transform yeast host cells, isolate positively transformed cells, and Extracting said second hybrid vector and said intracellular domain binding protein Applying yeast two-hybrid method to obtain sequence encoding protein to bind A method comprising:   (ix) a protein capable of binding to an intracellular domain of a TNF receptor or a FAS receptor A method of isolating and identifying a protein; using the sequence of the present invention or a portion thereof as a probe. CDNA or genomic D having at least partial homology to it Capture sequences from NA library The captured sequence is then amplified by PCR and cloned to produce the sequence of the present invention. A clone encoding a protein having at least partial homology to Non-stringent Southern hybridization followed by PCR A method consisting of applying a cloning method It is.   The present invention also provides a fine composition comprising any one of the following as an active ingredient: A pharmaceutical composition that regulates TNF or FAS ligand action on vesicles is there. That is, (i) the protein of the present invention or the proteins p55IC and p55 DD, FAS-IC or FAS-DD, biologically active fragments and analogs thereof , A derivative or a mixture thereof; (ii) a TNF receptor or a FAS receptor. Virus surface proteins that can bind to specific receptors on tumor cells or tumor cells. Recombinant animal virus vectors, and proteins, analogs, and Or a derivative or encodes p55IC, p55DD, FAS-IC or Is a sequence encoding FAS-DD; (iii) a virus surface tag as described in (ii) above. Recombinant animal virus vectors encoding proteins and p55IC, p55D D, which encodes the antisense sequence of the FAS-IC or FAS-DD sequence A lignonucleotide sequence; and (iv) a protein, analog or derivative of the invention. MRNA sequences encoding the body, or also p55IC, p55DD, FAS-IC Or a protein that can interact with the mRNA sequence encoding FAS-DD. This is a vector encoding a bozyme sequence.   A specific embodiment of the above aspect of the invention is p55-IC or encoding it The use of DNA. In this embodiment, p55-IC is linked to a ligand (TNF). Is a self-contained and independent TNF-associated effect in cells. It is based on the finding that it triggers. Therefore, TN in cells or tissues A method for inducing an F-related effect, comprising the steps of: There is provided a method comprising treating with a body or derivative. And the one or Are self-associating and independent of ligand (TNF), Can induce said TNF action at essentially all p55TNF receptors Essentially all or part of the body's self-associating intracellular domain (p55-IC) Selected from the group of proteins, wherein the treatment of the cells is performed by the one or more proteins. The protein, analog or derivative in a form suitable for introduction into the cell. Introducing into said cell, or said one or more proteins, analogs or Represents the DNA sequence encoding the derivative in the form of a suitable vector carrying said sequence. The vector is introduced into the cell. As presented, the sequence can be inserted into the cell.   An embodiment of the method of the present invention includes the following method. That is,   (i) the treatment of the cells comprises transfecting the cells with a recombinant animal virus vector; Project, and   (a) a window capable of binding to a specific cell surface receptor on the surface of the cell to be treated; Ruth surface protein (ligand) And, when expressed in the cell, self-associate with one or more of the P55-IC, a portion thereof, all of which can induce a TNF-related effect of Recombination carrying a second sequence encoding a protein that is an analog and derivative of all Constructing an animal virus vector; and   (b) infecting the cell with the vector of (a); A method consisting of:   (ii) the TNF action induced in the cell is induction of IL-8 gene expression. The vector comprises essentially all of p55-IC, a portion thereof, and the p55-IC. Carrying sequences encoding all analogs and derivatives of Signer that self-associates when expressed in cells and induces expression of the IL-8 gene The way in which the ring can be performed;   (iii) Treat tumor cells or virus-infected cells or antibacterial action of granulocytes A method of increasing the tumor cell; Virus Riga that can bind to specific cell surface receptors on the surface of stained cells or granulocytes And p55-IC, portions thereof, analogs thereof, and the like. And a sequence encoding the derivative and the protein, TNF, when expressed in stained or granulocyte cells, results in the death of these cells How to induce a related effect;   (iv) a method of treating a tumor cell; said p55-IC, a portion thereof, and the like. When the analog or derivative is expressed in tumor cells, it can induce granulocytes and other lymphocytes. IL-8 chemistry attracts to tumor cells and results in killing of the tumor cells A method of inducing the expression of IL-8 which leads to the death of said tumor cells by chemotactic activity; There is.   In this embodiment of the present invention, by inducing TNF-related effects intracellularly, The intracellular domain of the p55 receptor (p55-IC), used to treat cells And analogs and derivatives of all those mentioned above are provided. And its implementation The embodiments are as follows. Ie   (i) treating cells by inducing IL-8 gene expression in the cells; P55-IC, moieties, analogs and derivatives used for:   (ii) Inducing the expression of the IL-8 gene in the cell to cause the death of the tumor cell P55-IC, portions, analogs and derivatives used to treat tumor cells Body;   Further in this aspect of the invention, p55-IC, a portion thereof, all of the foregoing Contains analogs and derivatives as active ingredients and contains pharmaceutically acceptable carriers Composition for treating cells by inducing TNF-related effects in the cells It provides things. The embodiment is as follows. Ie   (i) as active ingredients, p55-IC, portions thereof, Derivatives and proteins that can bind to cell surface proteins on the cells to be treated Intracellular TNF-related effects comprising an encoding recombinant animal virus vector A pharmaceutical composition used to treat cells by expressing   (ii) administration induces IL-8 expression and kills tumor cells, A pharmaceutical composition used to treat tumor cells;   In yet another aspect, the present invention relates to a method for preparing at least two self-associated fusion proteins. Oligomeric tumor necrosis factor receptor (TNF receptor) comprising To offer. Then, each fusion protein has (a) TNF at one end thereof. TNF binding selected from the extracellular domain of the receptor, analogs or derivatives thereof Domain, wherein said extracellular domain, an analog or derivative thereof, Cannot bind and bind to TNF, and (b) At the other end, (i) the amino acid of the natural p55TNF receptor molecule (p55 receptor) Intracellular p55TNF receptor extending from about residue 206 to about amino acid residue 426 Essentially all of the domain (p55-IC); (ii) the amino acid of the natural p55 receptor Killed p55-IC extending from about residue 328 to about residue 426 Main; (iii) Books of intracellular domain of Fas / APO1 receptor (Fas-IC) Qualitatively all; (iv) the death domain of Fas-IC; and (v) self-association Selected from analogs, fractions or derivatives of any one of (i) to (iv) Having at least two self-associating domains; Said terminal (a) capable of binding to at least two TNF monomers, wherein said terminal (b) And each end (a) can bind to one TNF monomer. , TNF receptor, and salt and mechanism of the soluble oligomeric TNF receptor It is a functional derivative.   Embodiments of this aspect of the invention include combinations of terminal (a) and terminal (b) as defined above. All, for example, soluble Gomer's TNF receptor has a p55 receptor extracellular domain as the extracellular domain. And p55-IC as a self-associating intracellular domain.   Further, the present invention provides a method for producing the soluble oligomer TNF receptor of the present invention. Things.   Ie   (a) constructing an expression vector encoding any one of the fusion proteins, The DNA sequence at each end of the fusion protein is the extracellular region of the TNF receptor Almost all cloned encoding its analogs or derivatives From DNA sequence and almost all of said p55-IC, death of p55-IC Killing domain, Fas-IC, killing domain of Fas-IC, all of the foregoing Obtained from a cloned DNA sequence encoding an analog or derivative, The ends are joined to form a fusion protein sequence, and then the fusion protein Sequence is inserted into the vector under the control of transcriptional and translational regulatory sequences. Inserted;   (b) introducing the vector of (a) into a suitable host cell in which the fusion protein is expressed; Enter; then   (c) purifying the fusion protein expressed in the host cell, The quality is allowed to self-associate before, during or following the purification process to form soluble soluble A method comprising obtaining a Gomer's TNF receptor.   Further, a vector encoding the fusion protein useful in the method of the present invention. A host cell containing the vector; and, as an active ingredient, the soluble yeast of the present invention. Lignomer TNF receptor, salt or functional derivative thereof Containing any mixture of the above, and containing a pharmaceutically acceptable carrier A pharmaceutical composition is provided. Similarly, a soluble oligomeric TNF receptor of the invention, The salts, functional derivatives and mixtures of any of the foregoing are in particular excess T To treat conditions where NF is formed endogenously or administered exogenously Use it to mitigate the adverse effects of TNF in mammals, or use TNF When used exogenously, maintains the beneficial effects of TNF in mammals for extended periods of time Used to   In line with the concepts set forth with respect to the above aspects of the invention, the adverse effects of Fas ligand Soluble oligomeric Fas / APO1 receptor useful for attenuation (Fas receptor) It was discovered that it was possible to construct So in another way The present invention provides a soluble orifice containing at least two self-associated fusion proteins. Gomer's Fas / APO1 receptor (Fas receptor). Synthetic protein consists of (a) an extracellular domain of the Fas receptor, Similarity of the Fas receptor extracellular domain that cannot associate and can bind to Fas ligand And (b) at the other end, (i) native p55TNF From the amino acid residue at about position 206 to the amino acid at about position 426 of the receptor molecule (p55 receptor). Of the intracellular domain of the p55TNF receptor (pp-IC) extending to the acid residues (Ii) from amino acid residue at about position 328 to about 42 from the natural p55 receptor; A killing domain of p55-IC extending to amino acid residue position 6; (iii) Fas / A (Iv) Fas; essentially all of the intracellular domain of the PO1 receptor (Fas-IC); The killing domain of the IC; and (v) An analog or derivative of any one of (i) to (iv) capable of self-association Having at least two self-associating domains selected from: The protein is capable of binding to at least two Fas ligand monomers. self-associates only at the terminal (b) with (a), each terminal (a) And Functional Derivatives of Fas Receptors for Soluble Monomers and Soluble Oligomers Can be combined.   In this aspect of the invention, the soluble oligomer of the Fas receptor comprises the following steps: A manufacturing method is provided. Ie   (a) constructing an expression vector encoding any one of the fusion proteins, The DNA sequence at each end of the fusion protein is identical to the extracellular domain of Fas receptor. Clones that encode essentially all of the analogs or derivatives thereof. From the isolated DNA sequence and essentially all of the p55-IC Killing domain of Fas-IC, killing domain of Fas-IC, all of the foregoing Obtained from a cloned DNA sequence encoding all the analogs or derivatives, The ends are joined to form the sequence of the fusion protein, and the fusion protein Sequence is inserted into the vector under the control of transcriptional and translational regulatory sequences. Process;   (b) introducing the vector of (a) and an appropriate host cell in which the fusion protein is expressed; Entering; and   (c) purifying the fusion protein expressed in the host cell, and prior to the purification step, During or after the step, the fusion protein is self-assembled to form a soluble oligo. Ma Fa obtaining an s receptor; It is a method consisting of   Further, the present invention provides a soluble oligomeric Fas receptor useful in the method. Expression vector containing a fusion protein sequence to be loaded; a host containing the vector Cells; and as an active ingredient, a soluble oligomeric Fas receptor, or a salt thereof. Or a functional derivative or a mixture of any of the foregoing and is pharmaceutically acceptable. It is intended to provide a pharmaceutical composition which also contains an acceptable carrier. Similarly, soluble Rigomer Fas receptor, salt or functional derivative thereof or any of the foregoing Is provided, especially where excess Fas ligand is formed endogenously. Or treating exogenously administered conditions, the presence of Fas ligand in a mammal. Used to reduce harmful effects.   The same as described above for the oligomer TNF receptor and the oligomer FAS receptor. In the same way, a mixture having binding specificity for both TNF and FAS receptor ligand Combined oligomers can be produced. Thus, the present invention provides at least two Mixed oligomeric TNF receptor / FAS comprising self-associated fusion protein And one of the fusion proteins is one of the TNF-specific fusion proteins. And the other fusion protein is selected from the FA Selected from any one of the S receptor ligand specific fusion proteins, at least Also one TNF receptor extracellular domain and at least one FAS receptor extracellular domain. An intracellular domain that has a main and is fused to each of these extracellular domains. Kuha By binding the extracellular domain by self-associating between the parts A mixed oligomer is provided. These mixed oligomer acceptors, as described above, To produce oligomeric TNF receptor and oligomeric FAS receptor, Mix, then has binding specificity for both FAS receptor ligand and TNF Prepared by selecting standard oligomers by standard methods. The mixed oligomer Another method of producing the receptor is to use a TNF-specific fusion protein (as described above). Fusions encoding any of the soluble TNF receptors) and FAS receptor ligands Suitable vectors for vectors encoding any of the proteins (soluble FAS receptors) Co-transfect the main cells, purify and purify the expressed fusion protein Self-associate before, during or following purification to obtain the oligomeric receptor and then Oligomeric receptors capable of binding to both NF and FAS receptor ligands It is a method by selecting by the method.   Similarly, as the active ingredient, the mixed oligomer receptor, a salt thereof, or a functional inducer is used. Contains a conductor or a mixture of any of the foregoing and is pharmaceutically acceptable A pharmaceutical composition comprising a carrier is also provided. Further, the mixed oligomer receptor , A salt or derivative thereof or a mixture of any of the foregoing, in particular in excess TNF and FAS receptor ligands are formed endogenously or exogenously When treating a given condition, TNF and FAS receptor ligands in mammals Used to attenuate the adverse effects of both; or TNF and / or FAS receptor If gand (soluble form) is administered exogenously, Long-term beneficial effects of TNF and / or FAS receptor ligands in mammals Used to hold (slow release).   Other aspects and embodiments of the invention are provided in the following detailed description of the invention.   The following terms: "modulation of TNF action on cells" and "FA on cells" "Modulation of the action of S ligand", as used herein, refers to any in vivo treatment. It should be noted that this also includes in vitro treatment.                           BRIEF DESCRIPTION OF THE FIGURES   1a to 1c show that p55IC binding protein and p75IC binding protein Partial and preparatory nucleotide sequence (partial and pr eliminary nucleotide sequence) is schematically shown in FIG. FIG. 1 shows the sequence of clone 55.11 encoding fusion protein 55.11; b) Portion of clone 75.3 encoding p75IC binding protein 75.3 And FIG. 1 (c) shows the p75IC binding protein p75. The part of clone 75.16 encoding 16 and the preliminary sequence are shown Are all described in Example 1. FIG. 1 (d) shows the nucleus of FIG. 1 (a). The deduced amino acid sequence of protein 55.11 deduced from the nucleotide sequence is shown, Are also described in Example 1.   FIG. 2 shows that some cell lines were tested as described in Example 1. FIG. 4 is a copy of a Northern blot showing 55.11-specific mRNA present.   3A and 3B show that the protein encoded by the 55.11 cDNA is p55-I. Autora showing binding in vitro to a GST fusion protein containing part C FIG. 3A shows a copy of the full-length 55.11 protein (55.11 ful). l) binds to various GST fusion proteins, and FIG. The 55.11 portion fused to the AG octapeptide is various GST fusion proteins. Shows binding to quality. All of these are described in Example 1. .   FIG. 4 shows the predicted amino acid sequence of human 55.11 as described in Example 1. The results of comparing the columns to the sequence of the related protein from the lower organism are shown schematically.   FIG. 5 shows the results using an anti-MBP polyclonal antiserum showing self-association of p55IC. FIG. 4 is a copy of a stained western blot, wherein the western blot is Recently produced chimeric proteins p55IC-MBP and p55IC- GST (lanes 1-4) or chimeric protein p55IC-MBP and GST alone SDS-PA electrophoresed for German control interaction (lanes 5-8) Blots from GE gels, showing chimeric proteins (and controls) Interactions were performed prior to SDS-PAGE as described in Example 2. Performed on lutathione-agarose beads.   FIG. 6 shows transfected expression vector encoding full length p55IC. Cytotoxic effect of full-length p55IC in HT tal cells (right panel) This cytotoxic effect when treated with tracycline and blocking expression of the vector Phase contrast microscope showing inhibition (left panel) Dependence of transfected HeLa on cell death in cells Indicates no triggering. Ie   (i) At the left end of FIG. 7, HeLa cells were inserted into the transfected vector. , Full-length p55 receptor, its intracellular domain and parts of the intracellular domain Figure 2 schematically shows various DNA molecules encoding   (ii) Left and center bar graphs are HeLa cells of each type of receptor shown at the left end of FIG. Shows the expression of TNF receptor in cells, the left bar shows the amount of receptor (ng / cell sample) And the middle bar labeled with iodine bound to transfected cells The amount of receptor expressed by TNF is indicated.   (iii) The bar graph on the right shows the viability of HeLa cells expressing various receptors. Is shown.   In the bar graphs, open bars indicate transfer in the presence of tetracycline. The filled bars indicate the cells that were transferred and the filled bars show the transfer without tetracycline. Shows the cells that were cut. These are all described in Example 2 herein.   FIG. 8 shows translocation of the full-length p55 receptor or its intracellular domain (p55IC). Ligand-independent IL-8 gene expression in transfected HeLa cells Panel A shows the induction of HeLa cells with or without TNF. Northern blot showing the results of Northern analysis of RNA extracted from cells (“ Two lanes on the left, labeled "Control" and "TNF") and p55 Receptor, p55-IC or control He transfected with a vector encoding protein luciferase Duplicate Northern blot showing the results of Northern analysis of RNA extracted from La cells. Map (filled with “p55-IC”, “p55 receptor” and “Luc” respectively) The remaining lanes are shown, and the cells were treated with tetracycline in each case. (1 transfected) in the presence (+) or absence (-) of 2 lanes per injection). Panel B also includes HeLa shown in panel A. The results of staining 18S rRNA in each sample of cells with methylene blue are shown. This All of these are described in Example 2.   FIG. 9 (A and B) shows that the p55 receptor or a part thereof or FAS-IC Ligand-independent triggering of cell death in transfected HeLa cells The ring is shown graphically and FIG. 9A shows test results for the p55 receptor or a portion thereof. And FIG. 9B shows the test results for FAS-IC. FIG. 9A and FIG. The left panel of both 9B shows the p55 receptor used for transfection. Part or FAS-IC is schematically shown, and the right panel is a graph showing the experimental results. Indicated by All of these are described in Example 2.   FIG. 10 shows the portion of the cDNA clone designated F2 and the preliminary nucleoside. The nucleotide sequence is shown schematically and the nucleotide sequence is as described in Example 3. Encoding a protein capable of binding to p55IC and FAS-IC I have.   FIG. 11 shows the portion of the cDNA clone designated F9 and the preliminary nucleobase. Schematic representation of the tide sequence. The nucleotide sequence was p55IC and FAS-IC as described in Example 3. Encodes a protein that can bind to   FIG. 12 shows the portion of the cDNA clone designated DD11 and the reserve The leotide sequence is shown schematically and the nucleotide sequence is described in Example 3. Thus, a protein capable of binding to p55IC, especially p55DD and FAS-IC. It codes for Parkin.Detailed description of the invention   The invention relates in one aspect to a receptor belonging to the TNF / NGF superfamily, For example, it can bind to the intracellular domains of TNF and FAS receptors, Thus, for example, if TNF binds to the TNF receptor and the FAS ligand is FAS Plays a role in signaling processes initiated by binding to receptors The members of the superfamily, such as the TNF and FAS receptors; It relates to a novel protein that is considered a deleter or modulator. This Examples of these proteins include the intracellular domain of the p55TNF receptor (p55IC) , Such as 55.1, 55.3 and 55.1 herein. 1 (Example 1) and cDNA clones F2, F9 and And DD11-encoded proteins (Example 3); p75TNF receptor cells A protein that binds to the internal domain (p75IC), eg, 75.3 herein And a protein designated 75.16 (Example 1); Intracellular domain of the body (FAS-IC) , Such as the cDNA clones F2, F9 and DD11 Is a protein encoded by (Example 3). Proteins 55.1 and 55.3 , A portion or fragment of the intracellular domain of the p55TNF receptor (p55IC) It has been found that other proteins, 55.11, 75. 3 and 75.16 are proteins (7 5.3, 75.16) or was reported (55.11, Khan (1992)), its function and other properties, especially its ability to bind to TNF receptors. Is a protein that has never been reported (see Example 1 below). cD New proteins encoded by NA clones F2, F9 and DD11 These are completely unreported proteins, i.e. their sequences are DNA Or the amino acid sequence, “gene bank” or “protein bank (PROTEIN  BANK) "is not in the data bank.   Therefore, the present invention provides a DNA sequence encoding these proteins and It relates to the protein encoded by the sequence.   In addition, the present invention provides for the synthesis of biologically active analogs and derivatives of these proteins. DNA sequences and analogs and derivatives encoded by the DNA sequences . Such analogs and derivatives can be prepared using standard methods (eg, Sambrook). (1989)), which encodes these proteins. In the DNA sequence, one or more codons may be deleted, added or added to other codons. To the native protein Thus, an analog is obtained in which at least one amino acid residue has been changed. Acceptable similarity The body contains a TNF / NGF receptor superfamily, such as a FAS receptor or a TNF receptor. -Family intracellular domains, such as p55IC, p75IC or FA Analogs that retain at least the ability to bind S-IC; or other binding activity if Or analogs that can mediate enzymatic activity, such as p55, p75IC Or binds to FAS-IC but does not perform signaling, ie, further downstream Does not bind to a receptor, protein or other factor or produces a signal-dependent response An analog that does not catalyze. Thus, the so-called dominant negative effect (dominant-neg analogs having an ative effect), for example, p55IC, p75IC or In the binding to FAS-IC or signaling following this binding Defective analogs can be produced. Such analogs are, for example, By competing with the native IC binding protein, TNF or FAS- Can be used to inhibit the action of a ligand. Similarly, for example, if TNF Or the so-called dominant positive analogs (do Minant-postive analog) can also be manufactured. These analogs are Or better or better than the same IC binding properties and native IC binding proteins. It has excellent signaling characteristics. Similarly, derivatives are known in the art. As is known, the side groups of one or more amino acid residues of the protein are standardized. The protein and another molecule, such as an antibody, an enzyme By bonding to a receptor, etc. Can be manufactured.   These new TNF and FAS receptors bind to the intracellular domains. Proteins, such as 55.1, 55.3, 55.11, 75.3, 75.16. And the proteins encoded by the cDNA clones F2, F9 and DD11 ( F2, F9 and DD11) have a number of promising applications. And For example, the following uses.   (i) These proteins have high TNF or FAS receptor ligand action. Conditions, such as TNF or FAS receptor ligands In the case of anti-tumor, anti-inflammatory or anti-HIV uses where cytotoxicity is desirable For mimicking or enhancing the function of TNF or FAS receptor ligands it can. In this case, these proteins, for example, enhance the action of TNF. For example, 5.1, 55.3 and tandem binding to 55IC such as F2, F9 and DD11. Protein, as well as free p55IC itself (see below and Example 2) and p55 "Killing domain" of IC (p55DD); or do not act as FAS receptor ligand That is, proteins F2, F9 and DD11 which enhance cytotoxicity and FAS-IC And FAS-DD can be introduced into cells by standard methods known per se. for example For example, since these proteins are intracellular proteins, TNF or FAS Since it is desirable to introduce only into cells where the action of the receptor ligand is required, A system that specifically introduces these proteins into these cells is needed. this One way to do this is to use, for example, a recombinant animal virus derived from vaccinia virus. In this method, the following two genes are introduced into the DNA of this virus. So Genes are cell surface proteins specifically expressed by cells, such as AIDS (HI) that specifically binds to some cells (CD4 lymphocytes and related leukemias) V) a ligand that binds to a protein such as the viral gp120 protein, or Or a cell in which the recombinant viral vector carries a TNF receptor or a FAS receptor Specific for cells bearing TNF or FAS receptors so that they can bind to A gene encoding another ligand that binds to a protein; and a new intracellular domain Synthetic protein, ie, p55IC, p55DD, FAS-IC or FAS- It is a gene encoding a DD protein. Therefore, cell surface binding proteins When the protein is expressed on the surface of the virus, the virus is released from tumor cells or other TNF Targeting specifically to cells bearing the receptor or FAS receptor, Coding for an intracellular domain binding protein or p55IC, p55D The sequence encoding D, FAS-IC or FAS-DD is When introduced into cells and expressed intracellularly, TNF or FAS receptor ligands Tumor cells or other TNF receptors or FAS receptors that want to die due to increased action It causes the death of cells that hold the body. Construction of such recombinant animal viruses is standard (Eg, Sambrook et al. (1989)). Another possibility is the use of oligonucleotides that can be absorbed and expressed in cells. In the form, the new protein or p55IC, p55DD, FAS-IC Or those who introduce FAS-DD sequence There is a law.   (ii) these proteins are used, for example, in septic shock, graft versus host rejection TNF or FAS in cases such as reaction or tissue damage in acute hepatitis Used to inhibit the action of receptor ligands. In these cases, TN F induced TNF receptor intracellular signaling or FAS receptor ligand induced It is desirable to block outgoing FAS receptor intracellular signaling. In this case For example, sequences encoding antisense to these new proteins Or for p55IC, p55DD, FAS-IC or FAS-DD Oligonucleotides containing antisense sequences into cells by standard methods. And the mRNAs encoding these proteins Translation of TNF is effectively blocked, resulting in the blockade of the expression of that protein and TNF Inhibition of receptor ligand action or FAS receptor ligand action .   Such oligonucleotides are introduced into cells using the recombinant virus method described above. Wherein the second sequence that the virus conserves is the oligonucleotide Is an array. Another possibility is to use antibodies specific for these proteins Inhibiting their intracellular signaling activity. These new tampa Proteins can have extracellular domains as well as intracellular domains. , An intracellular domain that binds to the binding domain of a TNF receptor or a FAS receptor And using antibodies raised against their extracellular domains, their TNF is also Or FAS receptor ligands block related functions can do.   Still other methods of inhibiting the action of TNF or FAS receptor ligands are This is a recently developed ribozyme method. Ribozymes specifically open RNA A catalytic RNA molecule that cleaves. Ribozymes are selected target RNAs, such as MRNA encoding the novel protein of the invention or p55IC, p55D D, FAS-IC or FAS-DD Can be measured. Such ribozymes are specific for the selected mRNA And interact with the mRNA (complementary binding) to form the mRNA Cleavage to reduce (or completely eliminate) expression of the protein you want to inhibit And reduced levels of expression can be used to reduce the level of ribozyme expression in target cells. Is determined by Selected cells (eg, TNF receptor or FAS receptor In order to introduce ribozymes into cells that carry the body) Always used plasmids, animal virus (retrovirus) vectors Any suitable vector can be used (e.g., If the virus has the cDNA to be loaded as the second sequence, see (i) above. See). Furthermore, for example, the proteins of the invention and / or p55IC, p55 Used to inhibit the expression of one or more of DD, FAS-IC or FAS-DD To construct a ribozyme with multiple targets that can be used (multi-target ribozyme) (For a review of ribozymes and methods, see Chen et al. (1. 992), Zhao and Pick (1993) Shore et al. (1993), Joseph and Burke. (1993), Shimayama et al. (1993), Canto (Canto) r) et al. (1993), Barinaga (1993), Crise (ll) et al. (1993) and Koizumi et al. (1993)).   (iii) The new proteins of the present invention are other proteins, For example, cells located downstream of the intracellular domain of the TNF receptor or FAS receptor Isolate and identify other proteins involved in the intracellular signaling process and then clone Can be used to convert In this case, these options, The coding DNA sequence is used in a yeast two-hybrid system (see Example 1 below) In that system, the sequences of these proteins Proteins that bind to the intracellular domain of TNF or FAS receptors Other sequences encoding proteins that can bind ("preys") A or isolated from a genomic DNA library, cloned and then identified Used as “baits” to do so. Similarly, certain proteins of the invention Ie, proteins that bind to p55IC, p75IC or FAS-IC Can bind to other members of the TNF / NGF superfamily of receptors Or not. For example, p55 and p75 TNF It has recently been reported that there are other TNF receptors other than the body (Schwalb) et al. (1993); Baens et al. (1993); Crowe et al. (1994) Year)). Therefore, using the yeast two-hybrid system, various proteins of the present invention can be used. Quality of these other TNF receptors or other members of the TNF / NGF superfamily The ability to specifically bind to the receptor can be clearly tested. further, This method is based on the fact that the proteins of the present invention have other active and functional roles. Can be used to determine if it can bind to a receptor.   (iv) The new proteins of the present invention are other proteins of the same class, NF receptor or FAS receptor intracellular domain or functionally related receptor Isolate and identify proteins involved in intracellular signaling processes It can then be used for cloning. For this purpose, the yeast two hybrid Or non-stringent southern hybrids can be used. After performing non-stringent southern hybridization, PCR A recently developed cloning system (Wilks et al. (198) 9 years)). Wilkes et al. Published two putative proteins. Tyrosine kinase identification and cloning methods are described, Based on the known sequence of the motif, i.e., the devised kinase sequence. After performing stringent hybridization, clonin Is performed. This method uses the new protein sequence to Thus, related TNF receptors, FAS receptors or related receptors (TNF / NGF superfamily receptor) Can be used for cloning.   (v) Yet another method of utilizing the new proteins of the present invention is the novel Proteins or factors to which the new protein can bind, such as the TNF receptor (TNF / NGF receptor superfamily) related other receptors or cells Isolate and identify other proteins or factors involved in the intracellular signaling process Therefore, it is a method used for affinity chromatography. In this application The proteins of the present invention can be individually used as a substrate for affinity chromatography. And then extract the cells that are thought to be involved in the intracellular signaling process. Contact with the product or the isolated protein or factor. This affinity -After binding to the new protein of the present invention, Proteins or factors can be eluted, isolated and characterized .   (vi) As described above, the novel proteins of the present invention are used as immunogens (antigens). Alternatively, it can produce antibodies specific to it. These antibodies are used in cells New from extracts or from transformed cell lines producing the new proteins of the invention Can be used to purify new proteins. In addition, these antibodies Abnormal functioning of the TNF or FAS receptor ligand system, Induced by TNF or FAS receptor ligands that are excessive or too low Can be used to make a diagnosis confirming a disorder associated with the cellular action of Accordingly Dysfunctional intracellular signaling involving new proteins Such antibodies may serve as important diagnostic tools when related to the system .   The isolation, identification and characterization of the novel proteins of the present invention are well-known standard selection methods. It can be carried out using any of the above. For example, in one of these sorting methods Certain yeast two-hybrid methods described in the following examples (Examples 1 and 3) Was used to immobilize the new protein of the present invention. Mentioned above or below As can be seen, affinity chromatography, DNA known in the art Using other methods, such as hybridization methods, the new tanks of the present invention can be used. Isolate and identify and characterize the protein or use the novel proteins of the invention. Proteins that can bind to proteins or receptors belonging to the TNF / NGF family of receptors Isolate, identify and characterize additional proteins, factors, receptors, etc. Can be.   For the antibodies mentioned throughout this specification, the term "antibody" includes Riclonal antibody, monoclonal antibody (mAb), chimeric antibody, soluble type (soluble)  form) or antibodies that can be labeled in bound form Anti-idiotype (anti-Id) antibodies, as well as but not limited to enzyme cleavage Before being provided by any known method, such as a method, peptide synthesis or recombinant method Include fragments of the antibodies.   Polyclonal antibodies are heterologous forms of antibody molecules derived from the sera of animals immunized with the antigen. It is a group. Monoclonal antibodies form a substantially homogeneous population of antibodies specific for the antigen. And this population has substantially similar epitope binding sites. mAb It can be obtained by methods known to those skilled in the art (eg, Kohler And Milstein,Nature(Nature) 256 volumes, 495- 497, 1975; U.S. Patent No. 4,376,110; Ausubel) et al., Antibodies: Laboratory Manual A LABORATORY MANUAL "Cold Spring Harbor Laboratories Harlow of "Cold Spring Harbor Laboratory" (1988) And Lane, edited by Colligan et al., Current Protocols   Current Protocols in Immunology, Green Park Brishing Asok and Willei Interscience (Greene publi shing Assoc. and Wiley Interscience), New York (1992, 1995) Year). The contents of these cited references are entirely incorporated herein.) . The antibody is an immunoglobulin containing IgG, IgM, IgE, IgA, GILD. And its subclasses. Hive producing mAb of the invention Redomas can be cultured in vitro, in situ or in vivo . This is a currently preferred product because high titer mAbs are produced in vivo or in situ. Manufacturing method.   A chimeric antibody is a molecule whose different parts are derived from different animal species, For example, a molecule having a variable region derived from a mouse mAb and a constant region of a human immunoglobulin. is there. Chimeric antibodies primarily reduce immunogenicity during use and increase yield during production Used to let. For example, mouse mAb has a high yield from hybridomas. Higher but more immunogenic in humans, the human / mouse chimera m Ab is used. Chimeric antibodies and methods for their production are known in the art. (Cabilly et al., Proc Natul Acad Sai USA (P Roc. Natl. Acad. Sci. USA), 81, 3273-3277, 1984; Morrison et al., Proc Natul Acad Sai USA, Volume 81, 6851-6855, 1984; Bouulianne et al., Nature. (Nature), 312, 643-646, 1984; Cabilly Et al., European Patent Application No. 125023 (November 14, 1984) Published); Neuberger et al., Nature, 314, 2 68-270, 1985; Taniguchi et al., European Patent Application No. No. 171496 (published Feb. 19, 1985); Morrison ) Et al., European Patent Application No. 173494 (published March 5, 1986); Neuberger et al., PCT Patent Application No. WO8601533 Pamphlet (Published March 13, 1986); Kudo et al., European Patent Application No. 184187, published June 11, 1986; Sahaga n) et al., J. Immunol., 137, 1066-1074, 1986; Robinson et al., International Patent Application No. WO8702671 Unfret (released May 7, 1987); Liu et al., Procnatul   Acad Sai USA (Proc. Natl. Acad. Sci. USA), 84, 3439 ４３, p. 3443, 1987; Sun et al. USA (Proc.Nacl.Acad.S ci. USA), 84, 214-218, 1987; Better et al. Science, 240, 1044-1104, 1988; and c. Harlow and Lane, Antibodies: ANTIBODIES   Laboratory Manual (A LABORATORY MANUAL) (supra). These pulls The references are incorporated herein in their entirety.   Anti-idiotypic (anti-Id) antibodies are commonly associated with the antigen-binding site of an antibody. An antibody that recognizes a unique determinant. Id antibody , Animals of the same origin and species and genotype (eg, mouse strain) Prepared by immunizing with that mAb for which anti-Id has been prepared be able to. The immunized animal is used to determine the idiotype of the antibody to be immunized. By producing antibodies against the group (anti-Id antibodies), these idiotypes Recognize and respond to determinants (see, for example, US Pat. No. 4,699,880). , Which is hereby incorporated by reference in its entirety).   The anti-Id antibody may also be used as an "immunogen" to produce an immune response in yet another animal. To induce so-called anti-anti-Id antibodies. This anti-anti-Id The epitope is identical to the original mAb from which anti-Id was induced. Therefore By using an antibody against the idiotypic determinant of the mAb, Other clones expressing the antibodies of the invention can be identified.   Thus, the IC binding proteins of the invention, analogs or derivatives thereof, or even Or p55IC, p55DD, M generated for FAS-IC, FAS-DD, analogs or derivatives thereof Ab is used to induce anti-Id antibodies in suitable animals such as BALB / C mice. Can be used. Using spleen cells from such immunized mice, anti-IdmA b-secreting anti-Id hybridomas can be produced. Furthermore, this anti-Id The mAb is responsible for, for example, keyhole limpet hemocyanin (KLH). Can be conjugated to the body and used to immunize additional BALB / C mice You. The sera from these mice was the same as the IC-binding protein, its analogs. Or derivative, or p55IC, p55DD, FAS-IC or FAS-D D, the original mA specific for the epitope of the analogue or derivative thereof b) contains an anti-anti-Id antibody having the binding specificity of b.   Thus, the anti-Id mAb may have its own idiotype epitope or "Id." Which is similar in structure to the epitope being evaluated, such as GRB protein-α. Iotope ".   The term “antibody” also refers to intact molecules and antigens, such as Fabs And F (ab ')_TwoAnd both fragments of the complete molecule. Fab and F (Ab ')_TwoThe fragment lacks the Fc fragment of the intact antibody, circulation), and more specific than non-antibody Low in sex (Wahl et al., J. Nucl. Med., 24) Vol., 316-325, 1983).   Fab and F (ab ') of antibodies useful in the present invention_TwoAnd other fragments, Child disclosed herein According to the method, the IC binding protein or p55IC, p55DD, FAS -Can be used to detect and quantify IC or FAS-DD. Such a fragment Generally, papain (used to produce Fab fragments) or pepsin (F (ab ′)_TwoProteolytic cleavage method using an enzyme (such as used to produce fragments) Manufactured in.   An antibody can specifically react with a molecule and bind that molecule to that antibody If so, the antibody is said to be "bindable" to the molecule. "Epitope" The term is a molecule that an antibody can recognize and bind to. Means a part of An epitope or "antigenic determinant" is usually Consists of chemically active surface groupings of molecules such as amino acids or sugar side chains Have specific three-dimensional structure characteristics and specific charge characteristics.   An “antigen” is one that can be captured by an antibody and A molecule or portion of a molecule capable of causing an animal to produce an antibody capable of binding. An antigen has one or more epitopes. The specific response is highly advanced Reacts with its corresponding antibody in a manner that is selective for Not react with a number of other antibodies.   Antibodies (including antibody fragments) useful in the present invention include IC-binding proteins in samples. Or p55IC, p55DD, FAS-IC, FAS-DD quantitative or qualitative Or an IC-binding protein of the invention or p55IC, p55DD , FAS-IC and FAS-DD to detect the presence of cells expressing proteins Used to Can be This can be used for light microscopy, flow cytometry or fluorimetry. Immunofluorescence using antibodies labeled with fluorescence (see below). ).   Antibodies (or fragments thereof) useful in the present invention include the IC binding proteins or Performs the original position detection of p55IC, p55DD, FAS-IC and FAS-DD Therefore, it can be used histologically in immunofluorescence or immunoelectron microscopy. it can. In situ detection involves taking a histological sample from the patient and using the labeled antibody of the invention. Apply to the sample. The antibody (or fragment) is a labeled antibody (or fragment). ) Is preferably applied by coating or coating the biological sample. New By using said method, an IC binding protein or p55IC , P55DD, FAS-IC and FAS-DD, as well as Can be confirmed. Using the present invention, those skilled in the art will be able to Biological methods (eg, staining methods) can be improved to achieve such in situ detection Will be readily apparent.   An IC binding protein of the invention or p55IC, p55DD, FAS-IC, Assays such as FAS-DD are generally used for biological samples, such as biological fluids. Freshly harvested cells such as body, tissue extracts, lymphocytes or white blood cells, or Cells cultured in tissue culture can be purified using IC binding protein or p55IC, p55D. D, FAS-IC, detectably labeled antibody capable of identifying FAS-DD Incubate in the presence of the body and then by several methods known in the art. Detecting antibodies It is a method consisting of   Biological samples can immobilize cells, cell particles or soluble proteins. Solid support such as nitrocellulose or other solid support or carrier It can be treated with a carrier or carrier. The support or carrier is suitable. And then detectably labeled according to the invention as described above. Antibody. The solid support or carrier is then buffered. A second wash can be performed to remove unbound antibody. Next, the support Alternatively, the amount of the label bound to the carrier can be detected by an ordinary method.   "Solid support", "Solid support", "Solid support", "Solid support", "Support" Alternatively, the term "carrier" as used herein refers to an antigen or antibody binding. A compatible support or carrier is meant. With a known support or carrier Glass, polystyrene, polypropylene, polyethylene, dextran , Nylon, amylase, natural cellulose and modified cellulose (modified cellu) lose), polyacrylamides, gabbro and magnetite. The nature of the carrier It may be somewhat soluble or insoluble for the purposes of the present invention. Support Material is virtually possible as long as the bound molecule can bind to the antigen or antibody, etc. Any structure may be used. Therefore, the form of the support or carrier is Spherical, or on the inside surface of a test tube or the outside surface of a rod. May be cylindrical. Alternatively, use a sheet test strip (shee t test strip). Preferred supports or There are polystyrene beads as a carrier. One skilled in the art will recognize that Or knows many other suitable carriers for binding antigens, or Can be used for routine experimentation.   The binding activity of a given lot of the antibody of the present invention as described above is measured by a known method. can do. One of ordinary skill in the art will be able to determine each measurement using routine experimentation. Operating conditions and optimal test conditions can be determined.   Other steps such as washing, stirring, shaking, filtration etc. are customary for individual cases. It may be added to the assay either directly or as needed.   One method by which an antibody of the invention can be detectably labeled is to convert the antibody to an enzyme. This method is used for enzyme immunoassay (EIA). Next, the enzyme Subsequent exposure to a suitable substrate can be accomplished, for example, by spectrophotometry, fluorescence React with the substrate in such a way as to produce a chemical moiety that can be detected by the step. antibody Enzymes that can be used to detectably label Not included, malate dehydrogenase, staphylococcal nuclease, δ- 5-steroid isomerase, yeast alcohol dehydrogenase, α-glycero Phosphate dehydrogenase, triose phosphate isomerase, horseradish peroxy Sidase, alkaline phosphatase, asparaginase, glucose oxidase , Β-galactosidase, ribonuclease, urease, catalase, Lucose-6-phosphate dehydrogenase, glucoamylase and acetylcholine There is an esterase. Detection is a colorimetric method that uses a chromogenic substrate for the enzyme. Can be achieved. Detection was performed using a standard prepared in the same manner as the degree of enzymatic reaction of the substrate. It can be carried out by comparison with the standard and the visual observation.   Detection can be achieved using various other immunoassays. For example, antibodies Alternatively, radioimmunoassay (RIA) by labeling antibody fragments with radioactivity Can be used to detect RPTPase. Excellent theory about RIA Akira, TS (TS) et al., Edited by “Laboratory Techniques and Biochemistry in Molecular Biology (Laboratory Techniq ues and Biochemistry in Molecular Biology) ", North Holland Publi North Holland Publishing Company, New York, Especially during 1978, the title of Chard T. Reduction to radioimmune assay and relayed tech Neeks (An Introduction to Radioimmune Assay and Related Techniques) In the chapter. This document is incorporated herein by reference. Radioisotope The element is a method using a γ counter or a scintillation counter or It can be detected by autoradiography.   The antibody of the present invention can be labeled using a fluorescent compound. Fluorescently labeled When an antibody is exposed to light of the proper wavelength, its presence can be detected by fluorescence. Mo A commonly used fluorescent labeling compound is fluorescein isothiocyanate. G, rhodamine, phycoerythrin, picocyanine, allophycocyanin, o- Phthaldehyde And fluorescamine.   Antibodies are also of the lanthanod series.¹⁵²E using a fluorescent metal such as E It can be labeled for access. These metals are For example, diethylene tri Binding to antibodies using metal chelating groups such as amine pentaacetic acid (ETPA) Can be.   Antibodies are Label it detectably by attaching it to a chemiluminescent compound Can be attached. Next, The presence of an antibody labeled with chemiluminescence is a chemical reaction Is confirmed by detecting the presence of light emission that occurs during the process. Especially useful Examples of chemiluminescent labeling compounds are Luminor, Isorminol, Telomeric (th eromatic) acridinium ester, Imidazole, Acridinium salts Oxalic acid ester.   Similarly, The antibodies of the invention can be labeled using a bioluminescent compound. Bioluminescence is Catalytic proteins are found in biological systems that increase the efficiency of chemiluminescent reactions Is a kind of chemiluminescence. The presence of a bioluminescent protein can detect the presence of luminescence. And is confirmed by. Luciferin is an important bioluminescent compound used for labeling , Luciferase and aquorin.   The antibody molecules of the present invention may be used in a "two-site" assay or a "sandwich" assay. It can be employed for use in immunoassays known as routines. representative In a typical immunoassay, Solid support for a given amount of unlabeled antibody (or antibody fragment) Bound to a body or carrier and then added with a predetermined amount of a detectably labeled soluble antibody. In addition, Solid phase antibody, A ternary complex formed between the antigen and the labeled antibody Coalescence can be detected and / or quantified.   Typical preferred immunoassays are "normal" assays ("forward" assays). Yes, In this test, first, Contacting the antibody bound to the solid phase with the test sample, Futari By forming a solid phase antibody-antigen complex of the system, Remove the appropriate antigen from the sample After the incubation period, Washing the solid support or carrier, If exist If necessary, remove residues of fluid samples, including unreacted antigens, Then unknown quantities Contact with a solution containing a labeled antibody (which functions as a “receptor molecule”). Mark Intelligent antibodies, Complexes with antigen bound to solid support or carrier through unlabeled antibody After a second incubation period to allow coalescence, Its solid support or Performs a second wash of the carrier to remove unreacted labeled antibody.   Another type of "sandwich" test is Useful for the antigens of the invention , In this test method, a so-called "simultaneous" test method and an "inverse" test method are used. Simultaneous detection The usual method is The antibody bound to the solid support or carrier and the labeled antibody are both simultaneously This is a single incubation step added to the test sample. Incubate After completion, Wash solid support or carrier to form residue and complex with fluid sample Remove unlabeled antibody. Then bound to a solid support or carrier The presence of the labeled antibody Usually the same as for the "normal" sandwich test It is confirmed.   In the "inverse" test, First add a solution of the labeled antibody to the fluid sample and then add the appropriate After a period of cubation, Unlabeled antibody bound to a solid support or carrier Add Stepwise addition is performed. After the second incubation, Normal solid phase Washing is performed to remove the residue of the test sample and the solution of the unreacted labeled antibody. Then solid The labeled antibody bound to the support or carrier of "Simultaneous" and "normal" tests The measurement was performed in the same manner as in the case of the standard method.   For example, the yeast two hybrid method, Affinity chromatography and And any other standard selection method, such as methods known in the art. The novel protein of the present invention, which has been identified and characterized, Next, the standard recombinant DN A method (for example, see Sambrook et al. (1989)). . in this way, Proper eukaryotic or prokaryotic host cells, The protein of the present invention Transformed with a suitable eukaryotic or prokaryotic vector containing the sequence encoding Let Therefore, the present invention This is used to produce the proteins of the present invention. Expression vectors and transformed hosts. As mentioned above, these proteins Also include their biologically active analogs and derivatives, Therefore these tongues Vectors encoding protein include vectors encoding analogs of these proteins Is also included, Transformed hosts include those that produce such analogs. include. Derivatives of these proteins Produced by the transformed host A derivative produced by standard modification of the protein or its analog.   In another aspect, The present invention The free intracellular domain of the p55 TNR receptor (p55I C) or the free intracellular domain of the FAS receptor (FAS-IC) or Each so-called “dead domain” (p55DD or F AS-DD) on cells, Or its own TNF action or FAS receptor It relates to its use as a drug to enhance the ligand action (see Example 2). TNF again Indicates the cytotoxic effect induced by FAS receptor ligands, For example, cancer cells or HI If you want to introduce into cells like V-infected cells, p55IC, p55DD, FA S-IC or FAS-DD, The aforementioned recombinant animal virus (see (i) above) For example, it may be introduced into the cells using the vaccinia virus method. Also, Natural p55IC, p55DD, FAS-IC or FAS-DD, biological Analogs and derivatives or fragments that are active on can also be used, These are all It can be prepared as follows.   Similarly, the present invention provides p55IC, p55DD, FAS-IC or FAS-DD Of TNF action or FAS receptor ligand action by blocking the activity of Concerning the legal shutoff law, For example, introducing antisense oligonucleotides into cells p55IC, p55DD, Blocking the expression of FAS-IC or FAS-DD Can be.   The present invention also provides Intracellular domain binding protein of TNF receptor or FAS receptor Quality (p55IC, p55DD, FAS-IC and FAS-DD) A pharmaceutical composition comprising the recombinant animal virus vector to be loaded. And The vector is Can bind to specific target cell (eg, cancer cell) surface proteins Also encodes viral surface proteins, Intracellular domain binding protein in the cell Insert the protein sequence.   In another aspect, The present invention is particularly directed to the p55TNF receptor Self-associating intracellular domain (p55-IC, (See Embodiment 2). Kayo Examples of such effects are: An effect normally mediated by TNF binding to its receptor. And Similarly exerts signaling activity of self-associated p55-IC or a part thereof Action This is an action of inducing the expression of a gene encoding IL-8.   IL-8 belongs to a subclass of chemokine that has chemotactic activity. Is a cytokine that Other types associated with some pathological conditions, such as granulocytes Have been reported to play an important role in the chemotaxis of human cells (eg, Endo et al. (1994), Sekido et al. (1993), Harada ( Harada et al. (1993) and Ferrick et al. (1991)).   Since TNF has beneficial activity, By itself, Tumor cells and virus-infected cells It is used in treatments that destroy vesicles or increase the antimicrobial activity of granulocytes. But said like, TNF is Treatment of cancer, Used in large doses for antiviral or antibacterial treatment Including when you can, If you want to block that activity, ing.   Therefore, TNF or the beneficial activity of TNF can be exerted as well Substance It is particularly desirable to introduce the cells or tissues to be treated.   According to the present invention, Self-associating intracellular domain of p55 receptor (p55-IC) Can also exert some effects of TNF in a ligand-independent manner For example, the "killing domain" of p55-IC elicits cytotoxic effects on cells It is possible, p55-IC is IL-8 It has been found that gene expression can be induced. Therefore, using p55IC, To exert the function of TNF in a site-specific manner, In other words, P55-IC can be introduced only into cells or tissues.   One example of such a method is: Induces cytotoxic effects on tumor cells or malignant tissues Not only do Enhancing these effects by co-inducing IL-8 A DNA molecule encoding p55-IC or a part thereof, Is a method for specifically transfecting (transforming) tissues, Use this method Lymphocytes such as granulocytes accumulate at the site of the cells or tissues Helps destroy cells or tissues. This method has harmful side effects The need to perform large doses of TNF is avoided.   By using the usual recombinant DNA method, Study various areas of p55-IC Made Which regions are involved in each TNF-inducing effect You. For example, the present inventors have determined that the “killing domain” is involved in cytotoxicity. Confirmed, And various other constructs containing portions of p55-IC have already been prepared. , A part of this p55-IC is (Along with all or part of the dead domain) ) Involved in the action of other TNFs, Independent of ligands that are self-associating and active Used in the method These actions, For example, inducing an IL-8-inducing effect it can.   P55- involved in inducing other TNF-related effects (eg, IL-8-inducing effect) The sequence of IC It should be noted that the sequence differs from the sequence involved in cytotoxicity. And You That is, Contains no or only a small part of the "dead domain" Katsuso Have other sequence motifs from other regions of the intracellular domain of Or the same However, different characteristics of the same sequence (the same sequence motif) may cause other effects. Are involved.   Therefore, As mentioned earlier and described below, These p55-IC parts , An expression vector containing the analog or derivative thereof, Prepared, In the host cell To be expressed, Purified, It can be tested for its activity. in this way, One Or preparing some of the p55-IC fragments that have multiple TNF-related activities And Use in a specific way, For example, viral infections, Bacterial infections, Tumor Can treat any number of diseases. In all of these situations, Incorporate the p55-IC fragment involved in inducing IL-8 gene expression (or Co-transfection) can increase its specific activity, Hope Promotes destruction of cells or tissues to be destroyed by new IL-8 chemotactic activity be able to.   Therefore, Without administering TNF systemically, Cell or group to be treated In the weave, By specifically introducing all or part of p55-IC, A good effect can be induced.   p55-IC is obtained by any one of the above methods. The cells or tissues you want to destroy Can be specifically introduced. For example, One way to do this is Was For example, producing a recombinant animal virus derived from vaccinia virus, Of the virus Introduce the following two genes into DNA Is the way. The gene is A cell surface specifically expressed by the cell Protein, For example, specific for some cells (CD4 lymphocytes or related leukemia) Specifically binds to proteins such as the AIDS virus gp120 protein Ligand Or the TNF receptor preservation as in the recombinant virus vector Other resources that specifically bind to cells that have a TNF receptor capable of binding to cells A gene encoding Gand; And encodes p55-IC or a portion thereof Is a gene. Thus, cell surface binding proteins are expressed on the surface of the virus. When you let Virus specifically against tumor cells or other TNF receptor-bearing cells Pointing, Subsequently, the sequence encoding p55-IC or a part thereof Introduced into the cell. And when expressed in cells, The tumor cells you want to kill or TNF effects or death of other TNF receptor bearing cells For example, induction of IL-8 is increased. Such recombinant animal viruses are standard (Eg, Sam-brook et al. (1989)). Besides that, the sequence of p55-IC or a portion thereof Absorbed by cells and released inside cells It is also possible to introduce them in the form of oligonucleotides that can be expressed.   Therefore, the present invention encoding p55-IC or a portion thereof, Also A virus surface that can bind to surface proteins on specific target cells (eg, cancer cells) Also codes for proteins, The sequence of p55-IC or a portion thereof is inserted into the cell. And a pharmaceutical composition comprising said recombinant animal virus vector.   In another aspect, The present invention Be soluble and oligomerize to form dimers And can New synthetic TNF receptor that may form higher multimers About the condition. The molecules of the TNF receptor are such that the monomeric portion of each of these receptors is TNF Can bind to monomers. TNF is a homotrimer containing three active TNF monomers. Naturally exists as a mer, The monomers each bind to a single TNF receptor molecule Can be On the other hand, the TNF receptor exists as a monomer, The monomer is Each can only bind to one of the monomers of the homotrimeric molecule of TNF. Accordingly TNF Binds to three receptor molecules when binding to TNF receptor on cell surface So you can TNF receptors cluster. This means Signaling processes that ultimately trigger the effects of TNF observed on cells Is thought to be the beginning of   TNF is For example, it destroys tumor cells or virus-infected cells, Although having many desirable effects, such as the ability to increase bacterial activity, self Immune disorders, Rheumatoid arthritis, Graft-versus-host reaction (graft rejection), Septic show There are many undesirable effects, such as in the case of many serious illnesses, including shock. TNF is a major cause of pathological tissue destruction. TNF is also an activity of fat cells Suppressing causes excessive weight loss (cachexia). further, TNF is Its desired activity, such as when treating various malignant or viral diseases Even if administered for The dose of TNF used is Desirably Not some cytotoxic side effects, For example, causing destruction of healthy tissue in patients Though Often high enough.   Therefore, In all the above cases where the action of TNF is undesirable, With TNF Effective inhibitors have been sought. TNF binds to TNF and binds to its receptor Hinders the As a result, it contains soluble proteins that can inhibit the cytotoxic effects of TNF. Numerous TNF blocking agents have been proposed (EP 308378, EP39832 7 and EP568925). However, these TNF binding proteins or Since the soluble TNF receptor is a monomer, Each, TNF homotrimer TNF mono Can only bind to one of the markers. Therefore, the blockade of TNF function is not complete, Each TNF molecule to which a monomeric receptor has bound Binding to cell surface TNF receptor Two free T's that can illicit the action of TNF on cells It still has the NF monomer.   To overcome the drawbacks of blocking the function of TNF, Naturally occurring T A soluble male that can bind to at least two TNF monomers of the NF homotrimer molecule The present invention provides a method for constructing a ligomer TNF receptor as a fusion protein. It was developed. as a result, The TNF receptor of these soluble oligomers is More potent than known monomeric soluble TNF binding proteins or receptors Binds forcefully to its TNF ligand. For example, The soluble TNF receptor of the present invention In the case of dimer form, The receptor combines two TNF monomers of the TNF trimer. Because you can combine More complete neutralization of TNF occurs, In addition, the solubility of this dimer Because the dissociation rate of the body from TNF is slow, This neutralizing action Is It is a copy of the photo, This is described in Example 2.   FIG. Full-length p55 receptor, Its intracellular domain or “dead domain” Better persists in the portion of the intracellular domain that contains it. further, Such soluble ori Because the Gommer receptor is larger than that of its monomer, Clearance speed from body It is advantageous as a medicament because of its low potential.   The basis for the development of the soluble oligomeric TNF receptor of the present invention is is the p55 receptor The intracellular domain of the TNF receptor is capable of self-association, In addition, this intracellular domain There is a so-called “dead domain” in the main (p55-IC), This Can also self-merge, Cell to cell independent of ligand It was a discovery that toxic effects could occur (see Example 2). p55- Utilizing this self-association property of IC and its "dead domain" Standard recombination Using the DNA method, For example, p75 receptor or p55 receptor, Preferably p Almost all of the extracellular domains of the TNF receptor, such as the 55 receptor, Intracellular Substantially all of the death domain of the main (p55-IC) or p55-IC A fusion protein can be constructed by fusion. In this way, on the other hand A TNF binding domain at the end of That is, having the extracellular domain of the receptor, And Intracellular domain or self-associating domain that can self-associate to the other end A new fusion product having the formula Thus such a product is Two (Possibly more than two) self between p55-IC or its dead domain Oligomerize by association, At least two Obtain an oligomer having a TNF binding domain (ie at least a dimer) be able to.   further, Fas / APO1 receptor For p55-IC and its dead domain Having a certain homology, In self-associating cells containing self-associating "killing domains" It was discovered by the present invention that it had a domain (Example 2). Thus, the extracellular domain of the TNF receptor (as described above) Fas / APO1 By fusing to the intracellular or "dead" domain of the receptor, The soluble oligomeric TNF receptor of the present invention can be constructed.   In both cases, The oligomer TNF receptor of the present invention comprises: Possible TNF receptor A soluble extracellular domain; p55TNF receptor or Fas / AP01 receptor By having only the soluble intracellular domain or its killing domain It is soluble. That is, the oligomer TNF receptor of the present invention is a receptor for both types of Does not contain the body's transmembrane (insoluble) domain.   The construction of the oligomer TNF receptor of the present invention is described in detail in Example 4 below. State. But, When constructing the oligomeric TNF receptor of the invention, Ever reported Although not A situation has arisen in which the extracellular domain of the TNF receptor can self-associate. Note that this is the case. This state is Oligomer receptor is TNF Inhibit the ability of the motrimer molecule to bind to two or more TNF monomers, Also This is undesirable because the binding of such TNF monomers is not optimal.   Therefore, In such a case, Standard recombinant DNA Using the method For example, one or more amino acids contained within a self-associative region By deleting or substituting the acid residues, Extracellular TNF receptor The main can be modified to prevent such self-association. Therefore, TN Such modifications of the extracellular domain of the F receptor are part of the present invention, In this specification Denote analogs or derivatives of the extracellular domain of the TNF receptor. Similarly, Book Used for the oligomeric TNF receptor of the invention, p55 receptor or Fas / A PO1 receptor self-associating intracellular domain (IC) or its killing domain ( DD) An analogue or derivative thereof, That is, p55-IC sequence Or a modified version of that portion containing the killing domain (p55DD) or Fas / AP O1 intracellular domain (FAS-IC) sequence or dead domain (FAS DD) ). However, These modifications allow self-assembly A product can be obtained.   Similarly, A soluble oligomeric TNF receptor, An analog or derivative thereof Made After making and purifying, Further modified by standard chemical methods, These of the present invention Salt and machine for producing pharmaceutical composition containing TNF receptor as active ingredient Functional derivatives can be obtained.   To produce the soluble oligomeric TNF receptor of the present invention, TNF receptor The DNA sequence encoding the extracellular domain is Existing clones of all TNF receptors? Received Intracellular domain or killing domain and Fas / APO1 receptor Intracellular domain or killing domain can be obtained as it is (see Examples 2 and 5). See). This Thus, the DNA sequence of the desired extracellular domain is The desired intracellular domain containing the killing domain Main or Linked to the DNA sequence of that part, This fusion product then A suitable expression vector under the control of a promoter and other expression control sequences Inserted into (and linked to) Once the expression vector is formed, This is appropriate Introduced into host cells (transformation, Transfection), Next, To express Fusion of the invention is a soluble, self-associating TNF receptor molecule The product is obtained. These products are then purified from host cells by standard methods, The end product is a soluble oligomeric TNF receptor.   Preference is given to fusion products encoding extracellular and intracellular domains or parts thereof. A good manufacturing method is Copied from clones encoding all TNF receptor molecules PCR method using oligonucleotide specific to desired sequence It is. Other methods can be used, For example, extracellular domain and intracellular domain Isolating the desired portion of the encoding by restriction endonuclease; Next, the system These may be spliced together in a known manner with or without modification at the ends of the restriction fragments. Of the desired part of the receptor (extracellular and intracellular domains or parts thereof) There are ways to ensure accurate fusion. The fusion generation obtained in this way The product is inserted into the selected expression vector.   Similarly, The present invention Extracellular domain of Fas / APO1 receptor and p55 receptor The self-associating intracellular domain of the body (p55-IC), Its dead domain (p55 DD), Or the self-associating intracellular domain of the Fas / APO1 receptor (FAS-IC); Or its killing domain (FASDD) or an analog or derivative thereof (as described above). Oligomeric Fas / APO1 (FAS) receptor containing Related. The construction of these soluble oligomeric FAS receptors is described in detail in Example 5 below. To state, Sequences encoding available full-length cloned FAS receptors Is used as a starting material, PCR of desired extracellular domain and intracellular domain Using an appropriate oligonucleotide to produce in, Connect them to create a fusion Take a thing, The product is then inserted into a suitable expression vector. Earlier and As we will see later, Using prokaryotic or eukaryotic vectors and host cells Producing the desired soluble oligomeric FAS receptor, It is then purified and used as an active ingredient by a medical doctor. It is incorporated into a drug composition.   The soluble oligomeric FAS receptor of the present invention effectively blocks the Fas ligand. The purpose is to refuse, Because it exists as a trimmer (TNF alike, See above), Each oligomer receptor of the present invention, Two or two To neutralize its activity by binding to Fas ligand, which may exceed You. Although Fas ligand is known to bind predominantly to the cell surface, soluble Exists in type. in any case, The oligomeric FAS receptor of the present invention Because it is possible to bind to at least two monomers of the S receptor), The activity of Fas ligand can be effectively neutralized. Fas Activation of the ligand and the FAS receptor by this ligand Several pathological conditions state, Pathologies associated with liver damage, including liver damage associated with hepatitis, especially Self-infection, including lymphocyte damage (cell apoptosis) in humans infected with HIV Involved in immune conditions (eg, Ogasawara et al. (1993) ; Cheng et al. (1994)). Therefore, Soluble oligomer of the present invention The FAS receptor of In order to block the activity of Fas ligand, The above Pharmaceutical compositions for use in treating pathological conditions associated with such Fas ligands. It can be used as an active ingredient.   Similarly, The present invention Binding affinity for both TNF and FAS receptor ligands Of Soluble Oligomers with Tee or So-called "Mixed" TNF Receptors / FAS receptor oligomer receptor. These mixed oligomer receptors are: Small At least one TNF receptor extracellular domain and at least one FAS receptor cell Contains extracellular domains, Each of these extracellular domains Said self-associating p55 IC, p55DD, Fused to either FAS IC or FAS DD To form an oligomer receptor.   These mixed oligomers can be produced as follows. That is, (a ) Any one of p55IC and FAS IC in the self-associating intracellular region, Or self Either p55DD or FASDD of the self-association killing domain or its own Part of self-association, TNF receptors fused to either analogs or derivatives ( Extracellular domain of p75TNF receptor or preferably p55TNF receptor) Providing any of the above fusion products containing And; (B) self-associating p55IC, FAS-IC, p55DD and FAS Any one of DD or a self-associating portion thereof; Either analog or derivative Any of the above fusion products containing the extracellular domain of the FAS receptor fused to Provide them; (C) Any of the TNF-specific fusion products of (a) A) mixing with any of the FAS receptor ligand-specific fusion products of at least Containing both the TNF receptor and the extracellular domain of the FAS receptor, These domains Are bound by the self-association performance of the IC or DD region fused to it. Oligomer (dimer or higher oligomer) receptor (standard selection) ・ This is a method that is provided)   Other methods by which the mixed oligomer acceptor can be prepared include: Suitable for the above expression vectors By co-transformation of an intact host cell, One of the vectors is Encoding a TNF-specific TNF receptor fusion product, Another vector is FAS It encodes a receptor ligand-specific FAS receptor fusion product. These different After expressing the fusion product in a host cell, Mix by standard purification A ligomer (TNF receptor / FAS receptor) receptor is obtained.   The effectiveness of these mixed affinity oligomer receptors is Mainly TNF And FAS receptor ligands are endogenously overexpressed or exogenously administered To neutralize both when the levels are undesirably high is there. According to recent evidence, FAS receptor ligand (usually, On the cell surface ) And TNF- A possible synergistic effect between α (also on the cell surface) It is pointed out that there is a possibility. Therefore, In some cases, Both these Riga It is desirable to neutralize the antibody at the same location on the cell surface. That is, such a mixture The affinity receptor is TNF binding to its receptor and FAS receptor ligand Can block both binding to the receptor. Therefore, this The mixed affinity receptor Expected to work both FAS receptor ligands in TNF Effective for pharmaceutical compositions used to treat undesirable conditions (see above) Can be used as a component.   Similarly, TNF receptor, FAS receptor and mixtures of soluble oligomers of the invention Along the directions described above for the TNF receptor / FAS receptor oligomer, other Receptors or mixtures thereof, In particular, other members of the TNF / NGF superfamily Can produce soluble oligomeric receptors for any of the bar receptors You. In this case, One of the extracellular domains of various receptors, The self-association property In the intracellular region or its part, Or the supermarket that can hold a self-meeting Can be fused to any other intracellular domain of any of the family members You.   Expression of any of the recombinant proteins of the invention described herein is Appropriate expression Eukaryotic cells (eg, yeast, Insect cells or mammalian cells) Can do it. Methods known in the art can be used.   For example, DNA molecule encoding a protein obtained by any of the above methods Is Methods known in the art Can be inserted into an expression vector appropriately constructed in (Sambrook (Sambbrook) rook) et al. (1989)). Double-stranded cDNA is In the plasmid vector, Homopo Homopolymeric tailing or synthetic DNA linkers Alternatively, the ligation is performed by a restriction ligation method using a blunt end ligation method. DNA ligase Ligating the DNA molecules using Undesired bonds are treated with alkaline phosphatase. To avoid it.   In order to express the desired protein, The expression vector is Gene expression Ligation to DNA encoding desired protein in such a way that protein can be produced Done, Contains specific nucleotide sequences with information that regulates transcription and translation Must be there. Primarily, The gene is To be transcribed, RNA polymerase Must be preceded by a recognizable promoter, The promoter has R NA polymerase binds and initiates the transcription process. Used by various promoters Has been Works with different efficiencies (strong and weak promoters There is). Promoters differ between prokaryotic and eukaryotic cells.   Examples of the promoter that can be used in the present invention include: All configuration promoters - Bacteriophage ΔintPromoter, β- of pBR322 Lactamase geneblaPromoter and chlorampheny of pPR325 Such as the CAT promoter of the call acetyl transferase gene, Are the major promoters of the inducible promoter, for example, right and left of bacteriophage Δ. Tar (P_LAnd P_R), E. colitrp,r ecA ,lacZ,lacJ,ompFandgalPromoters ortrp-lac There are prokaryotic promoters, including hybrid promoters ( Glick B.R. (1987)). Generates large amounts of mRNA High levels in prokaryotic cells in addition to using strong promoters to Ribosome binding sites for efficient mRNA translation to achieve gene expression It is necessary to make sure the translation is done. One example is the placement of a suitable position from the start codon. Shine-Dalgarno sequence that is complementary to the 3'-terminal sequence of 16S RNA ( SD sequence).   In the case of eukaryotic hosts, transcriptional and translational regulatory sequences (regu latory sequewe) is used. These regulatory sequences have high levels of regulatory signals. Adenovirus, bovine nipple when linked to a specific gene that expresses Sequences from the origin of the virus, such as oncovirus, simian virus, etc. Examples include the herpes virus TK promoter, the SV40 early promoter, And the yeast gal4 gene promoter. As a transcription initiation regulatory signal, Those that can suppress and activate so as to regulate the expression of genes are selected.   D containing a nucleotide sequence encoding the protein of the fusion product of the invention An NA sequence is operably linked to allow integration of a desired gene sequence into a host cell. Inserted into a vector that has the transcriptional and translational regulatory signals in place. Introduced The cells that have been stably transformed with the modified DNA are isolated from host cells containing the expression vector. Introducing one or more markers that can select cells Therefore, it can be selected. Markers for phototrophy An auxotrophic host may be exposed to biocides such as antibiotics or heavy metals such as copper. Provides resistance to killing agents. The selectable marker gene is the DNA to be expressed Can be ligated directly to gene sequence or transfected in the same cell Can be introduced by Additional for optimal synthesis of proteins of the invention Element is required. These elements include transcription promoters, enhancers , And a termination signal. A cDNA expression vector incorporating such elements Okayama, H. (1983) reports that Have been.   In a preferred embodiment, the introduced DNA molecule undergoes autonomous replication in the recipient host. Incorporated into a plasmid or viral vector that can be transferred. Specific programs An important factor when choosing a rasmid or viral vector is the choice of vector. Receptor cells containing the vector are recognized and selected from recipient cells not containing the vector. Ease of separation; the number of copies of the vector required in a particular host; Whether it would be desirable for a vector to be able to "shuttle" between certain host cells That is.   Preferred prokaryotic vectors are plasmids that can replicate in E. coli, For example, pBR322, ColE1, pSC101, pACYC184, etc. Maniatis et al. (1982) and Sambrook et al. (1989)); Bacillus such as pC194, pC221, pT127, etc. Plasmid (Gryczan, T. (1982) p. Streptomyces plasmid containing LI101 (Kedal KJ ndall, K.J.) et al. (1987)). Lyophage "Sixth International Symposium on Acti Nominetals Biology (Sixth International Symposium on Actinomycet ales Biology ”(1986) by Chater, K.F. ); And a Pseudomonas plasmid (John, J.F.) (1986) and Izaki, K. (1978). Good Preferred eukaryotic plasmids include BPV, vaccinia, SV40, And the like or derivatives thereof. Such plasmids are publicly available in the art. (Botsteim, D. et al. (1982); "The Molecular Biology of the East Saccharomyces: Life Cycle and Inheritance (The Molecular Biology of the Yeast Sac charomyces: Life Cycle and Inheritance) (1981), Brooch Broach, J.R .; Brooch, JR (19) (1982); Bollon, D.P. et al. (1980); Iodology: A Comprehensive Treatise (Cell Biology: A Compreh ensive Treatise) Vol.3 Gene Expression ”(Gene Expression) 1980), Maniatis, T .; and Sambrook, S. ambrook) et al. (1989).   A vector or DNA sequence containing the construct is generated. Once prepared for actual use, the DNA construct can be prepared by any suitable method, Transformation, transfection, conjugation, protoplast fusion, electroporation, Appropriate host cells can be obtained by either calcium phosphate precipitation or direct microinjection. Can be introduced into cells.   The host cell used in the present invention may be a prokaryotic cell or a eukaryotic cell. Preferred prokaryotic host E. coli, Bacillus, Streptomyces (Streptomyces) ), Pseudomonas, Salmonella, Sera There are bacteria such as the chia (Serratia). The most preferred prokaryotic host is E. coli. A particularly important bacterial host is E. coli K12 strain 294 (ATCC 31446). Enterobacteriaceae X1776 (ATCC 31537), E. coli W3110 (F^-, Λ^-, Hara Sakae Nutrition (ATCC 27325)) and Salmonella typhimurium (Salm onella typhimurium or Serratia marcescen intestinal bacteria such as s) and bacteria of various species of the genus Pseudomonas It is. Under such conditions, the protein is not glycosylated. Prokaryotic host Must be compatible with the replicon and control sequences in the expression plasmid.   Preferred eukaryotic hosts are mammalian cells, such as humans, monkeys, mice and tea cells. Inneys hamster ovary (CHO) cells. Because these hosts, Protein molecules, including correct folding or glycosylation at the correct site After the translation. Yeast cells are translated, including glycosylation Subsequent peptide modifications can be performed. Desired protein in yeast Strong promoter sequences and high copy number plasmids that can be used to produce There are several recombinant DNA strategies using Yeast is a cloned mammal Recognize the leader sequence of a class of gene products and use the peptide (leading That is, it secretes a pre-peptide.   After the introduction of the vector, the host cells are expanded by selecting cells containing the vector. Grow in selective medium to be grown. Expression of the cloned gene sequence The desired protein is produced.   Purification of the recombinant protein may be performed by any one of the known methods for this purpose. Is done. That is, it includes extraction, sedimentation, chromatography, electrophoresis, etc. This is the usual method. It is used preferentially to purify the protein of the present invention. Subsequent purification is performed by affinity chromatography using an anti-TNF receptor monoclonal antibody. It is matography. In addition, this antibody is generated on the gel base material contained in the column. And fixed. Impurity-containing preparation containing recombinant protein is loaded onto the column Let it pass. The protein is captured on the column by a specific antibody while the impurities Passes. After washing, the protein may change pH or ionic strength To elute from the gel.   The term “salts”, as used herein (see above), refers to The salts of the carboxyl group of the protein of the present invention formed with known methods in the field Both acid addition salts of a mino group are meant. As a salt of a carboxyl group, for example, Like sodium salt, calcium salt Inorganic salts and salts such as triethanolamine, arginine or lysine Salts with organic bases, such as those formed with mins. Examples of acid addition salts include minerals There are salts with acids and salts with organic acids.   The term "functional derivative" as used herein refers to a residue or N-terminal Alternatively, a method known in the art from a functional group present as a side chain of a C-terminal group Which are still pharmaceutically acceptable That is, it does not destroy the activity of the protein and makes the composition containing it toxic. Unless given, it is included in the present invention. These derivatives include carboxyl groups Aliphatic esters or amides, and acyl moieties (eg, alkanoyl groups Or carbocyclic aroyl group) to form an O-acyl derivative of a free hydroxyl. There are N-acyl derivatives of free amino groups on conductors.   The term "fraction", as used herein, may refer to a portion of the receptor. Or part (intracellular or extracellular domain of the receptor) or intracellular of the receptor A part or part of a protein that binds to a domain. But the tongue Parkin retains its biological activity.   As described above, the present invention relates to pharmaceutically acceptable carriers, and various described inventions. Including the active ingredient or its salts, functional derivatives or mixtures thereof Various pharmaceutical compositions. These compositions may be in any of the states described herein. For example, septic shock, cachexia, graft-versus-host reaction In response, endogenous TNF such as cases of autoimmune symptoms such as rheumatoid arthritis Some cases are overproduced. The administration method is Any of the accepted modes of administration for similar drugs, depending on the condition being treated. Round. For example, if the composition of the present invention is used to inhibit TNF action, sepsis Administered intravenously for cases of sexual shock or local for cases of rheumatoid arthritis Administered by local injection (for example, into the lap) or continuously by infusion You. The compositions of the invention may also be used, for example, in the treatment of cancer or viral diseases. Case of TNF poisoning caused by exogenous administration of TNF in excess (overdose) Can also be used.   The pharmaceutical composition of the present invention physiologically permits the protein of the present invention or a derivative thereof. Manufactured for administration by mixing with acceptable carriers, stabilizers and additives. And in a dosage form, for example by lyophilization in a dosage vial bottle. Built. The amount of active compound administered will vary with the route of administration, the disease being treated and the patient being treated. Depends on symptoms. For example, in the case of cases of inflammatory symptoms of rheumatoid arthritis, Intravenous injection is more effective on a weight basis than intravenous injection in cases of septic shock. It is necessary to reduce the amount of minutes.   Other aspects of the invention will be apparent from the following examples.   The present invention will be described in more detail with reference to the following examples and the accompanying drawings. The drawings do not limit the invention.Example 1 Cloning of proteins that bind to the intracellular domains of p55 and p75 TNF receptors Cleaning and isolation   Intracellular domain of p55 and p75 TNF receptors Interacting with (p55IC and p75IC) To isolate yeast, the yeast two-hybrid syste m) was used (Fields and Song, 1989).   Briefly, the two-hybrid system comprises a DNA binding domain and an activation domain. Eukaryotic organisms, such as GAL4, with two distinct domains, main Specific in vivo by repair of transcriptional activator Yeast-based genes for detecting complex protein-protein interactions Analysis. The domain comprises a repaired GAL4 protein expressed and bound to each other. Once formed, it can bind to upstream activating sequences and then, for example, La A report whose expression, such as cZ or HIS3, is observed in readily cultured cells Activates the promoter that controls the expression of the reporter gene You. In this system, candidate interacting protein genes are expressed separately. Cloned with the current vector. In one expression vector, one candidate protein The protein sequence is cloned simultaneously with the sequence of the domain that binds to GAL4 DNA. To produce a hybrid protein with the GAL4 DNA binding domain, In the other vector, the sequence of the second candidate protein is a GAL4 activated domain. Cloned with the GAL4 activation domain and hybridized with the GAL4 activation domain. To produce protein. Next, the two hybrid vectors are Enzyme having lacZ or HIS3 reporter gene under control of L4 binding site Co-transformed into the mother host strain. Two hybrids Transformed host cells capable of expressing and interacting with each other Only (co-transformants) will be able to express the reporter gene . In the case of the lacZ reporter gene, the host cell expressing this gene is X-ga 1 will appear blue when added to the culture. Therefore, a blue colony Allows two cloned candidate proteins to interact with each other Shows the fact that   Using this two-hybrid system, the intracellular domains p55IC and p7 5IC was cloned into the vector pGBT9 (clone carrying the binding sequence of GAL4 DNA). Available from CLONTECH, USA. See below) separately To prepare a fusion protein having the DNA binding domain of GAL4 (the same as above). Thus, a portion of the intracellular domains FAS-IC and 55IC, ie, 55 DD is also cloned into pGBT9 to isolate other IC binding proteins Used for. See Example 3 below). pGB for p55IC and p75IC For cloning into T9, the p55TNF receptor (Schall et al., 1990) and the p75TNF receptor (Smith et al., 1990). Using a clone encoding the full-length cDNA sequence, intracellular domain The in (IC) was cut out. p55IC using the enzymes EcoRI and SalI And the EcoRI-SalI fragment containing the p55IC sequence was ligated to the standard Isolated by the procedure, and the multiple cloning site region was identified using EcoRI and SalI. Into the pGBT9 vector opened in the region (MCS) Was. p75IC was excised with the enzymes BspHI and SalI, followed by p7 The BspHI-SalI fragment containing the 5IC sequence was isolated by standard procedures and Filled-in with Nou enzyme, opened with SmaI and SalI A fragment that could be inserted into the pGBT9 vector was generated.   Next, the above-mentioned hybrid (chimeric) vector was inserted into the activation domain of GAL4. C derived from human HeLa cells cloned into a pGADGH vector HF7c yeast host strain was co-transfected with the DNA library (separate In each case, one cotransfection was performed with the p55IC hybrid vector. Alternatively, the other co-transfection with p75IC hybrid vector (Performed) (all vectors described above, ie pGBT9 and HeLa cells) PGADGH and an enzyme strain having a cDNA library of MATCHMAKER Two-Hybrid System, #PT Clontech Laborat as part of 1265-I ories. Inc.), purchased from the United States. ). Cotransfected yeast The mother was treated with a histidine-deficient medium (His)^-Medium)). (Growing colonies indicate positive transformants). Then selected Yeast clones are distinguished by their ability to express the lacZ gene, ie, their L Tested for ACZ activity. This test is based on the yeast encoded by the lacZ gene. Metabolized by elemental β-galactosidase to form a blue product X-gal as culture medium This was done by addition. Therefore, the blue colonies are active lacZ Genes can be indicated. GAL4 transcriptional activity for lacZ gene activity That the factor is present in an active form in the transformed clone, ie GAL4 DNA binding domain encoded by the hybrid vector, GAL4 activation domain encoded by the other hybrid vector and appropriate It is necessary to combine Such a combination is The two proteins fused to the four domains interact with each other stably (binding Yes) is only possible. Therefore, the isolated His⁺And And blue (LACZ⁺) Colonies are harbored by a vector encoding p55IC and p. Protein production from human HeLa cells that can stably bind to 55IC Colonies co-transfected with the vector encoding the product, or p It is capable of stably binding to a vector encoding 75IC and p75IC. Transfection with a vector encoding a protein product of human HeLa cell origin This is the colony that is being controlled.   The His⁺, LACZ⁺Isolate plasmid DNA from yeast colonies and Electroporation into E. coli HB101 strain by a typical procedure, followed by Leu⁺ And ampicillin resistant transformants (these transformants were Amp^RAnd Le u^TwoA hybrid pGADGH vector having both of the sequences encoding That is what you want). Therefore, such transformants are p55IC or Or a novel that can bind to p75IC Is a coulomb having a sequence encoding the protein identified in (1). Then plastic Isolating the Smid DNA from the transformed E. coli, (A) replacing the plasmid with the original intracellular domain hybrid plasmid ( Hybrid pGTB having either one of the p55IC or p75IC sequences The yeast HF7 strain described above together with 9) is transformed again. As a control, Vectors having sequences encoding unrelated proteins, such as pACT- Lamin (pACT-lamin) or pGBT9 alone can be used for p55IC binding protein or Is used for co-transformation with a plasmid encoding the p75 IC binding protein. You. The co-transformed yeast was then His^-Medium alone or different levels of 3- His containing aminotriazole^-Test for growth on medium; and And (B) the plasmid DNA, the original intracellular domain hybrid plus Plasmid and the control plasmid described in (a) were used as the yeast strain SFY526. Transform into mother host cells, LACZ⁺Activity (the efficiency of β-gal formation, ie blue Color) Was retested.   According to the results of the above test, His^-Colony growth patterns in culture Is identical to the pattern of LACZ activity as assessed by Ronnie's color, ie His⁺Colony is LACZ⁺It turned out that there was. In addition, liquid culture ( LACZ activity under preferred culture conditions), GAL4 DNA binding and activation Domain hybrids with higher GAL4 than HF-7 yeast host cells Transfection into SFY526 yeast host capable of inducing LACZ by transcriptional activator I evaluated it after the effect.   The results of the co-transfection are shown in Table 1 below. From the above results, That many proteins can bind to p55IC or p75IC, A protein named 55.11 that binds p55IC, p75IC 75.3 and 75.16, which bind to All p55I C and p75IC binding proteins were used in the yeast two-hybrid analysis system. Fused to the GAL4 activation domain sequence of plasmid pGADGH all encoded by cDNA sequences from a cell cDNA library It is a genuine human protein.   Interestingly, fragments of p55IC itself, namely 55.1 and 55.3, We have also found that the named protein can bind to p55IC. These are also discussed in Example 2 below.   In Table 1 above, the plasmid and the GAL4 DNA binding domain and G Hybrids encoding the AL4 activation domain are as follows.DNA binding domain hybrid pGBT9-IC55: the intracellular domain of the full-length p55-TNF receptor (p55 IC) pACT-lamin: an unrelated protein, lamin pGBT9: vector alone pGBT9-IC75: the intracellular domain of the full-length p75-TNF receptor (p75 IC)Activation domain hybrid   55.1 and 55.3 bind to fragments of the intracellular domain of the p55-TNF receptor. Respond.   55.11 is a novel protein that associates with the p55-TNF receptor.   75.3 and 75.16 are novel proteins that associate with the p75-TNF receptor Quality.   The novel p55IC binding protein and p75IC binding protein 55.11 and 75.3 and 75.16, For the cloned cDNA, sequence using standard DNA sequencing procedures. It was determined. The partial sequences of the sequences encoding all these proteins are shown in FIGS. 1 (a) shows the sequence of the cDNA encoding protein 55.11. FIG. 1 (b) shows a partial sequence of cDNA encoding protein 75.3. FIG. 1 (c) shows a partial sequence of cDNA encoding protein 75.16. ing. FIG. 1 (d) shows protein 5 deduced from the nucleotide sequence of FIG. 1 (a). The deduced amino acid sequence of 5.11 is shown.   However, the partial sequence of the cDNA encoding the 55.11 protein is: Khan et al. (1992) Year) also determined human brain cDNA sequencing and physical and genetic mapping. Brain cDNA directed at establishing a new rapid and accurate method for performing It should be noted that this has been reported in studies of the sequence of However, Kahn et al. Describes the function of the protein encoded by the sequence of the 55.11 cDNA or It does not provide any information about other properties and No other analysis is included in the work of Khan et al.Analysis and characterization of 55.11 protein (A) General procedures and materials (i) Cloning of 55.11 cDNA   Analysis of cDNA for protein 55.11 (eg Northern analysis, see below) 55, cloned by the two hybrid screen procedure . Eleven cDNAs encode amino acids 309-900 (see FIG. 1 (d)). 55.11 partial having nucleotides 925-2863 (see FIG. 1 (a)) It was revealed that only the cDNA was represented. 55.11 of the cDNA Nucleotides 1 to 3 encoding the remaining part (amino acids 1 to 308 (FIG. 1 (d))) 924 (FIG. 1 (a))), using the standard procedure, ie, live human fetal liver cDNA. Rallyed by PCR (see below for details) . The full nucleotide sequence of 55.11 (FIG. 1 (a)) was Both by the chain termination method Direction.(ii) Two-hybrid β-galactosidase expression test   The β-galactosidase expression test was performed in several of the aforementioned tests to determine the A pVP16 vector containing an activation domain is transformed into a Gal4 activation domain vector. The procedure was as described above, except that a certain pGAD-GH was used instead of pGAD-GH. Was. The numbering of residues in the protein encoded by the cDNA insert is Same as in the Swiss-Prot data bank It is. Deletion mutants are produced by PCR and point mutations are directed to oligonucleotides It was produced by mutagenesis (oligonucleotide-directed mutagenesis). Kunkel, 1994).(iii) Northern analysis   Total RNA for TRI REAGENT (Molecular Research   Center (Molecular Research Center, INC.), Cincinnati, Ohio (Oh.), United States) and formaldehyde / formamide Denatured in buffer, electrophoresed on agarose / formaldehyde gel, Genescreen Plus Men in 10X SSPE buffer using standard techniques Blen (Gene Screen Plus membrane) (Dupont, Wilmint) , De., USA). The blot was 55.1 1 with the partial cDNA (see above, nucleotides 925-2863) Let soybeans, random-prime kit (Boehringer)   Mannheim Biochemica, Mannheim Radioactively labeled (Mennheim, Germany) and stringently ) Washed. Autoradiography was performed over a week.(iv) Expression of 55.11 cDNA in HeLa cells and     And p55-IC glutachi of 55.11 protein     On-S-transferase fusion protein binding     Combination   Glutathione S-transferase (GST) fusion with p55-IC (GST-p55IC) and truncated downstream from amino acid 345. A fusion with p55-IC (GST-p55IC345) was produced and Adsorbed to glutathione-agarose beads as described in Example 2 (Smith (Smith) and Corcoran, 1994; Frangio ni) and Neel, 1993). 55.11 cDNA (1-2 863 nucleotides, ie 55.11 full-length cDNA), FLAG-55 . 11 cDNA and luciferase cDNA were expressed in HeLa cells. Was. FLAG-55.11 contains 309-900 of the 55.11 protein. The region extending between residues (two hybrid screen 55.11 partial cDNA (nucleotide 925-2863)) and FLAG octapeptide (Eastman Koda Eastman Kodak Company, New Haven, City (ct.), United States Country) via N. Expression of the fusion protein is performed using tetracycline. Transactivator (tetracycline-controlled transactivat) or) expressing H Expression vector controlled by tetracycline in eLa cell clone (Tetracycline-controlled expression vector) (HtTA-1) (Example 2 below and Gossen and Bujard) , 1992). [³⁵S] methionine and [³⁵S] Cysteine (Dupont Inc., Wilmington, DE, United States and Amersham ), Buckinghamshire, UK) using metabolic labeling of expressed proteins ( metabolic labeling), lysis of HeLa cells, immunoprecipitation and labeled proteins Binding to quality GST fusion protein is 0% instead of 0.1% in cell lysis buffer. . The following (implemented) except that 5% Nonidet P-40 was present. The procedure was as described in Example 2). Immunization of 55.11 and FLAG-55.11 Sedimentation is a GS containing a 55.11 region extending between amino acids 309-900. Rabbit antiserum to T fusion protein (diluted 1: 500) and FLAG Mouse monoclonal antibody (M2, Eastman Kodak (5 μg / ml in cell lysate).(B) Binding of 55.11 protein to p55-IC in transformed yeast   In this study, we characterized the binding between 55.11 and p55IC, especially It is necessary to identify the regions of both these proteins involved in this binding. I was For this purpose, various full lengths in the "DNA binding domain" construct P55IC and deletion mutants of p55IC (see also Example 2 below) Tar GAL Sequence of 55.11 partial in 4AD and VP16AD (residues 309-900) , Isolated independently) is partially encoded by the construct fused to the "activation domain" "Baits" that bind to "preys", which is 55.11 protein The two-hybrid procedure used above was used. In addition, various 55.11 A deletion mutant was constructed and fused with the "activation domain" in the GAL4AD vector (Eg, 55.11 with only residues 309-680 and 457-900) Variants). Various "activation domain" constructs of various "binding domain" constructs Binding was tested in transfected SFY526 yeast cells. The binding Was evaluated by a two-hybrid β-galactosidase expression filter assay. Valued. Unrelated proteins, SNF1 and SNF4, are each labeled as Positive controls for the "main" and "activation domain" constructs Empty Gal4 (pGAD-GH) and VP16 (pVP 16) Vector as negative control for "activation domain" construct The empty Gal4 (pGBT9) vector into the "binding domain" construct Used as a negative control. The results of the analysis are shown in Table 2 below. , The symbols “++” and “++” in Table 2 are 20 to 60 after the start of analysis, respectively. Intense color development within minutes (positive binding result), "-" within 24 hours after the start of analysis Indicates no color development (negative result). Blank columns in the table indicate that binding analysis has not been performed Is shown.   From the results shown in Table 2 above, 55.11 corresponds to the “dead domain” of p55-IC. (Death domein) ”(residues 328 to 426) at a site different from p55-IC. Can be concluded.   55.11 protein kills more efficiently than uncleaved p55-IC Truncated p55-IC with the deletion domain deleted (construct 20 in Table 2) 6-328). In addition, a construct having a further truncated C-terminus (construct 20 6-308) and a construction in which the killing domain and the portion close to the membrane are both deleted. (Constructs 243-328). However, 55.11 protein The quality did not bind to the construct truncated from the N-terminus to amino acid 266 (Table 1). 2). Based on these findings, the binding site for 55.11 is a residue of p55-IC Located in the region extending between 243-308, the N-terminus of this binding site is at residue 2 43 to 266 are shown.   Unique cloning of 55.11 cDNA containing Gal4 activation domain From the "prey" construct to the play construct containing the VP16 activation domain As a result, the efficiency of binding of the 55.11 protein to p55-IC did not decrease. (Table 2). Therefore, one or more structures involved in this bond are 55.11 Belongs to the molecule and does not appear to be related to the fusion site with the 55.11 activation domain. Will be   However, the binding of 55.11 to p55-IC is due to its C-terminal (55.1). 1 constructs 309-680) or N-terminal (55.11 constructs 457-900). Izu It was abolished even with limited truncation in it (residue 309 was a two- Partial cDNA clones uniquely isolated in the hybrid screen Is the first residue in the 55.11 protein encoded by).   55.11 are three receptors of the TNF / NGF receptor family (p75 receptor Body, Fas / APO1 and CD40) and eg lamin and cyclin Contains other proteins such as cyclin D (data not shown) Of p55-IC, since it did not bind to Binding appears to be specific. Human FAS receptor (residues 175-319) ), CD40 (residues 216-277) and the p75-TNF receptor (residues 287 461), other TNF / NGF receptor proteins tested here The portion containing the intracellular domain was also examined, but none bound to 55.11 ( It should be noted that the data is not shown).(C) R from several cell lines using 55.11 cDNA as probe Northern analysis of NA and cloning of full length 55.11 cDNA   The cell line tested was HeL from human epithelial carcinoma. a cell, acute lymphoblastic T cell leukemi a) CEM cells derived from Jurkat cells from acute T-cell leukemia And HepG2 cells derived from hepatocellular carcinoma. The uniquely isolated cDNA of 55.11 (nucleotides 925-286) 3) was used as a probe. Is the sample 10μg RNA / lane It was composed of The results of the Northern analysis are shown in FIG. Show.   Therefore, FIG. 2 shows that the cDNA using 55.11 cDNA was used as a probe. Zan analysis showed that in some cell lines, the uniquely isolated 55.11 cD A single hybrid of approximately 3 kB, larger than the cDNA which is NA (2 kB) It is clear that a single hybridizing transcript was specified. PCR using oligonucleotide primers corresponding to the sequence of 55.11 A 5'extending sequence having a length of about 1 kB Cloned. This 5 ', which has the length of the independently cloned cDNA, The total length of the stretched sequence is close to the transcript length of 55.11. These parts and The 3 kB cDNA contained in the transfected HeLa cells Expressed (see below), producing a protein of about 84 kDa, which is 3 kB Suggest that the cDNA contains a translation initiation site.(D) GST containing the p55-IC portion of the 55.11 protein in vitro To fusion proteins   We confirmed that 55.11 could actually bind to p55-IC, To eliminate yeast protein involvement in binding, bacterially produced GST p5 5-IC fusion protein and 3k produced by transfected HeLa cells Phase with the protein encoded by the 55.11 cDNA of B (55.11-full length) The interaction was studied. In this study, a total length of 55.11, FLAG -55.11 (5 encoded by an independently cloned partial cDNA) 5.11 residues 309-900, fused to FLAG octapeptide at N-terminus He) transfected with luciferase (control) Expressed in La cells, [³⁵S] methionine and [³⁵S] Using cysteine Metabolically labeled. The following proteins were fused to GST: full length p55-IC (GST-p55-IC) and the C-terminus to amino acid 345 P55-IC (GST-p55-IC) from which most of "main" (see Table 2) has been removed. 345). GST alone was used as a control. Transfected cells Lysate was used to bind the full-length 55.11 protein to the GST fusion protein. Used for the 55.11 protein or FLAG When the -55.11 fusion product was used for binding to a GST fusion protein, FL Immunoprecipitation was performed using an antibody against the AG octapeptide. The protein is S DS-polyacrylamide gel electrophoresis (SDS-PAGE: 10% acrylic Mid), followed by autoradiography.   FIG. 3A and FIG. 3A showing the autoradiographic reproduction of the SDS-PAGE gel. 3A and 3B, FIG. 3A shows the full-length 55.11 protein (55.11-full length). FIG. 3B shows binding to various GST fusion proteins and FIG. Figure 4 shows binding of the combined products to various GST fusion proteins. In FIG. 3A In the rightmost lane, only the full length 55.11 was transfected and the anti-55 . 11 antibody (α55.11 antibody) Control immunoprecipitation of lysates of the precipitated cells is shown. In FIG. 3B In the lane of the rightmost band, only the FLAG-55.11 Infected and immunoprecipitated with an anti-FLAG antibody (αFLAG antibody). A lysate control immunoprecipitation is shown.   Thus, from FIGS. 3A and 3B, the full length 55.11 cDNA The expressed protein can be expressed in HeLa cells and has the full-length p55-IC Fusion protein containing GST-p55IC or lacking most of the killing domain A fusion protein containing cleaved p55-IC and (GST-p55IC345 ) It is clear that they bind (FIG. 3A). The full length 55.11 protein is GS It did not bind to T alone (control). Similarly, click at the beginning of 55.11. FLAG octapepti encoded by the cloned partial cDNA Protein (FLAG-55.11) expressed in HeLa cells fused with Bound with GST-p55IC and GST-p55IC345 in vitro , GST (FIG. 3B). Therefore, the above result indicates that 55.11 is " Region upstream of the p55IC of the "dead domain", ie, the transmembrane domain (Transmembrane domain) (See (b) above).   In addition, the study has shown that in accordance with the present invention, antibodies against 55.11 have been successfully The production is explained (FIG. 3A).(E) deduced amino acid sequence of human 55.11 and related tannins present in lower organisms Comparison of the deduced amino acid sequence of the protein with the sequence of the 55.11 protein Features of   As described above, in accordance with the present invention, a full-length 55.11 cDNA is cloned. And sequenced (see nucleotide sequence in FIG. 1 (a)). . 11 was deduced from the cDNA sequence (the amino acid sequence in FIG. Acid sequence). Data Bank (Genbank (TM) / EMBL Data Bank (GenBank^TM/ EMBL DataBank) search revealed a cDNA sequence of human 55.11. The column parts (Accession numbers T03659, Z19559 and F09128) and Homologues (accession numbers X80422 and Z31147) ) Is for arbitrary sequencing of cDNA libraries. It has become clear that it has already been decided. Culture of human hepatoma HC10 cells CDNA sequence encoding a 596 amino acid human protein present in nutrients ( Accession number U18247) is similar to the cDNA sequence of 55.11. But However, this hepatoma protein has an N-terminal corresponding to the N-terminal part of 55.11. Lacking the portion (amino acids 1-297), residues 297-377 at 55.11 and And 55.11 in the region corresponding to residues 648-668. data Bank search revealed that proteins with very high sequence homology to 55.11 The quality is Saccharomyces celebsier (Saccharomyces cerevisiae) (Yeast), a Rabidopsis Sariana (Arabidopsis thaliana) (Plant) and Caenorhub Ditis Elegance (Caenorhabditis elegans) (Worms) Was. Therefore, 55.11 appears to perform an evolutionarily conserved function. It is. In yeast, two DNA sequences resembling the 55.11 DNA sequence Known proteins (open reading frame for bases YHR027c and And SEN3) are present. Both proteins are 55.11 in size near. YHR027c is known only by sequencing genomic clones, On the other hand, SEN3 has been cloned as cDNA. 55.11 and YH Similarities between R027c are much higher than those between 55.11 and SEN3 It is clear that the site in 55.11 is similar to the site in SEN3 The site correlates with a site of 55.11 similarity to YHR027c. You. Arabidopsis Sariana and Kaenorhab, though only part Based on DNA sequence information obtained from Ditis elegans proteins, These proteins are similar to 55.11 as well as the mother YHR027c protein Is clearly shown. Among these four proteins, The only protein characterized has limited homology to 55.11 Yeast SEN3. SEN3 is a 20S proteasome (20Sproteasom) e) identified as the yeast equivalent of the p112 subunit of the activator (Proteolytic core of the 26S proteasome (Rextainer (Rech Steiner et al., 1993 and DeMartino et al., 1994)) ( M. Calvertso C. Culbertson and M. Hockstrasser , Personal information).   FIG. 4 shows the deduced amino acid sequence of human 55.11 and the amino acid sequence present in the lower organism. A schematic comparison with related proteins is shown. Sequence compared in FIG. Is 55.11 cDNA (see FIG. 1 (d)), 8 Open reading frame (YHR027c) for a nucleotide in the cosmid derived from the chromosome (nuclease) Otide 21253-24234, accession number U10399); SEN3, Saccharo CDNA of a protein of Mrs. celebsie (Accession No. L06321); A partial cDNA of the protein of R. rhidopsis sariana (accession number T2150) 0) and the partial c of Caenorhabditis elegans protein Amino acid sequence predicted for DNA (Accession number D27396). 55 . The "KEKE" sequence at 11 is marked with a solid line and the sequence AYAGS (x)₈ LL is marked by a dashed line. GCG package (CGCG package) PILEUP and PRETTYBOX)  programs). Maximize alignments Gaps introduced for this purpose are indicated by dashes.   Various sequence features or motifs (motifs) present in the human 55.11 sequence With regard to ()), the following was confirmed. The conserved amino acid sequence motif is Repeated "KEKE" spreading between lysine 614 and glutamic acid 632 Except for the sequence (underlined in FIG. 4), the protein encoded by 55.11 Admitted in quality I couldn't. Proteasonal subunits and chape The “KEKE” sequence present in a number of proteins including ronins (chaperonins) Arrays may facilitate the association of protein complexes (Reaiini et al.) , 1994). Array AYAGS (x)₈LL is in the 55.11 protein Appear twice (sites 479, 590; see FIG. 4), but the functional Meaning has not been explained yet.(F) Sequence features of the p55IC region involved in binding to the 55.11 protein   As described above (see (b) and (d)), the 55.11 protein is p5 The region between residues 243 and 308 of the 5-IC (the N-terminus of this binding site is residue 24 3 and 266) and this region is upstream of the “dead domain” And closer to the transmembrane domain of the p55-TNF receptor. 5 This region in p55-IC, to which 5.11 binds, has a high content of proline, serine And a threonine residue. However, this area has some RPM1 and RPM2 proline-rich genes present in several cytokine receptors Contains no chief (RPM1 and RPM2 proline-rich motifs) (O'Neill (O ' Neal) and Yu-Lee, 1993). The deletion causes the p55 receptor Region extending between residues 243-266, where binding to 55.11 is abolished (See (b) and (d) above and Table 2). Two threonines are followed by proline residues, which link these residues to the MAP kinase. , CDC2 and other proline dependent Site that is likely to be phosphorylated by existing kinases (Seger) And Krebs, 1995). Phosphorylation of this site in the receptor May affect its binding to the 55.11 protein.   All of the preceding statements regarding protein 55.11 and its binding to p55-IC. In view of the above, according to the present invention, a different upstream of the “dead domain” of p55-IC Can be concluded that a novel protein that binds to a region . Such binding may be due to TNF-mediated activity other than inducing cell death. ctivities). 55.11 joins The region is involved in the induction of nitric oxide synthase. Has already been shown (Tartaglia et al., 1993), and TNF Of sphingomyelinase (neutral sphingomyelinase) (Wiegmann et al., 1994). Accordingly In association with the 55.11 p55-TNF receptor intracellular domain (p55IC). The union (binding) is based on (i) signaling (s) for the above or other effects of TNF. ignaling), (ii) protein folding or processing (Suggested by similarities to subunits of the 55.11 26S proteasome. Or (iii) affect the regulation of p55-TNF receptor activity or expression. Or may be involved.Example 2 of the intracellular domain of the p55TNF receptor (p55IC) Self-association ability, ability to cause cell death, other features and activities of p55IC Sex and related intracellular domain of Fas / APO1 receptor   As shown in Example 1 above, the intracellular domain of the p55TNF receptor (p5 5IC) is capable of binding itself, and also fragments of p55IC, That is, proteins 55.1 and 55.3 can also bind to p55IC. I found out.   Binding of TNF to the p55TNF receptor causes cell destruction in cells having this receptor. It is known to lead to cytocidal effects. In addition, cells of this receptor Antibodies against the ectodomain can be used to crosslink the receptor ), The antibody itself can cause this effect.   In addition, mutation studies (Tartaglia et al., (1993), Brakebusch et al., (1992)) reported that the p55 receptor Its function depends on the integrity of the intracellular domain of the p55 receptor. Was shown. Therefore, the onset of signaling for the cytotoxic effect of TNF is Of two or more p55 receptors forced by receptor aggregation It was suggested to occur as a result of the association of the intravesicular domain (p55-IC). Book The result according to the invention is that the expression of the intracellular domain of the p55 receptor This finding indicates that cell death occurs without a membrane or intracellular domain. Provides further evidence for a solution. P55 receptor without such an intracellular domain The condition is likely to function independently of TNF It is shown to have a self-association that leads to the ability to be able to. Full-length p55 receptor That the signaling by TNF depends on stimulation of TNF reduces this self-association Or reflects the activity of the transmembrane or extracellular domain of the interfering receptor It is suggested that   The ability of the intracellular domain of the p55 receptor (p55-IC) to self-associate Attempts to clone effector proteins that interact with the receptor (see above) Fortunately, it was found in Example 1). For that purpose, the " -Hybrid technology. found to associate with p55IC In addition to the novel protein 55.11, the intracellular domain of the p55 receptor The cDNA sequence coding for the portion of is cloned into three other cloned HeLa cells. Implicates that the vesicle cDNA contains and that p55-IC can self-associate I found that. Two of these clones are identical and have amino acids 328 (P55IC protein fragment 55. Clone 55.1 which encodes No. 1). The third one is amino acid 27 It contained a long insert encoding 7-426 (protein fragment of p55IC). Clone 55.3 encoding 55.3).   In addition, two chimeric p55ICs produced by bacteria, one of which is maltaux Fused to G-binding protein (MBP) and the other is glutathione-S-tra To evaluate the in vitro interaction with the fusion with transferase (GST) Was. These chimeras are constructed, cloned, and expressed using standard methods. I let it. It Following their expression, GST-, a chimeric protein produced by the bacterium, IC55 (Mr-51 kD) and MBP-IC55 (Mr-67 kD) Of the p55 receptor intracellular domain (p55IC) Self-interaction was evaluated. Equivalent amount of GST-IC55 chimera (Samples of lanes 1 to 4 in FIG. 5) or GST alone (FIG. 5). Samples from lanes 5 to 8) were glutathione-agarose beads (Sigma). (Sigma) and then an equivalent amount of M in one of the buffer solutions shown below. Incubated with the BP-IC55 fusion protein.   (i) Buffer I (20 mM Tris-HCl, pH 7.5, 100 mM KC 1, 2 mM CaCl_Two, 2 mM MgCl_Two, 5 mM DTT, 0.2% tiger Triton X100, 0.5 mM PMSF, 5% glycerol Le). This buffer was used for the samples in lanes 1 and 5 in FIG.   (ii) MgCl_TwoBuffer I containing 5 mM EDTA instead of. This buffer is Used for 5 lanes 2 and 6 samples.   (iii) Buffer I containing 250 mM KCl instead of 100 mM KCl. This Was used for the samples in lanes 3 and 7 of FIG.   (iv) Buffer I containing 400 mM KCl instead of 100 mM KCl. this Buffer was used for the samples in lanes 4 and 8 of FIG.   Incubated at 4 ° C for 2 hours with rotation Afterwards, the beads are washed with the same buffer and boiled in SDS-PAGE buffer, Then, electrophoresis was performed by PAGE. Next, nitro the protein on the gel. Western blot on cellulose membrane, then polyclonal anti-MBP Staining was performed using serum. A copy of this stained Western blot is shown in FIG. The samples in lanes 1 to 8 are as described above.   From FIG. 5, it can be seen that the p55IC-MBP chimera was significantly independent of the divalent cation. Even under a high salt concentration (0.4 M KCl), the p55IC-GST chimera It is evident that it binds to (1) to (4). Therefore, p55IC actively (Avidly) It is concluded that they can self-associate.   To assess the functional relationship of p55-IC's tendency to self-associate, An attempt was made to express p55-IC in the cytoplasm of cells susceptible to vesicle disruption. Recently developed, taking into account that p55-IC may change to cytotoxic. A tightly regulated tetracycline-controlled mammalian expression system (te tracycline-controlled mammalian expression system) (Gossen and Boujard, 199). 2 years). Expression of p55-IC resulted in massive cell death (FIG. 6, Right panel). Dead cells are cells that are seen when cells are killed by TNF. Bubbling of the cell surface was indicated. Reportedly pHD Expression of 10-3 regulated constructs^FiveTetracycline reduces by a factor of two P55-IC structure in the presence of Transfection of architectural cells in the absence of tetracycline Although considerably lower than what was seen, it resulted in some cell death. (FIG. 6, left panel). In contrast, contains luciferase cDNA Cells transfected with the control construct showed no signs of death ( Results are not shown).   P when expressed without the transmembrane or extracellular domain of the receptor The ability of 55-IC to cause cell death depends on this domain in signaling. Provide further evidence of the relationship. In addition, the rest of the receptor It does not play a direct role in such signaling. The present invention Of the effects of mutations, including the mutations studied in Studies have shown that the region extending between amino acid residues 326-407 is responsible for this function. Was shown to be the most important. This region contains the p55TNF receptor Signals for two other evolutionarily related receptors, cell death Fas receptor (Itoh et al., 1991 and Oeh m) et al., 1992) and CD40 (Steme), a receptor that enhances cell proliferation. (Stamenkovic et al., 1989). It shows significant similarity (resemblance) and therefore this sequence It is thought to constitute a conserved motif that plays a role in some general way. Because it does not resemble the characteristics of known motifs of enzymatic activity, indirect methods Or perhaps signaling enzymes Or a docking site for a protein that transmits a stimulus signal to the enzyme ( It is plausible to signal by using it as a docking site) Will be p55-IC, Fas receptor and CD40 are all Can be stimulated by antibodies to the main. These stimuli are Can be shown to be related to the ability of the antibody to cross-link. Hence, Signalin The two or more intracellular cells forced by the aggregation of extracellular domains It is likely to begin as a result of domain interactions. These receptors For the relationship of such interactions in the initiation of gnarling, see the intracellular domain. Expression of a receptor that has become inoperable due to mutations in the Shows a "dominant negative" effect on the functioning of the body Study (Brakebusch et al., 1992). . Aggregation of the p55 receptor in response to TNF occurs as homotrimers. Each of the resulting TNF molecules is capable of binding two or three receptor molecules Only by the fact that it can happen in a passive manner It was suggested. However, in the knowledge of the present invention, this process is somewhat different. Suggests that   The tendency of p55-IC to self-associate is that this domain is involved in its induced aggregation. And plays a role of activation. Furthermore, p55-IC is a receptor When expressed independently of the rest of the molecule, TNF or any other external In the absence of external stimuli Can cause cell death at p55-IC, so that the activity of p55-IC Seems to be enough to get started. Nevertheless, it is expressed as a full-length receptor When the p55-TNF receptor is not signaled by TNF, Do not Therefore, when activating the p55-TNF receptor, TNF Suppresses certain inhibitory mechanisms that prevent spontaneous association of the inner domains, and this inhibition is It has been postulated that the IC is due to linkage to the rest of the receptor molecule. This inhibition Compulsory to intracellular domain by transmembrane and extracellular domain Orientation for the association of some other proteins with receptors n), or perhaps just a limitation on the amount of receptor allowed to be located at the cell membrane It might be because of that.   In order to cause cell death, even a single TNF molecule binds to the cell. Note that this control mechanism is quite effective, You have to watch.   Spontaneous signaling independent of ligand results in this receptor Can cause a great deal of derangement in the process of being controlled it can. The best-known example is growth factor receptor deregulation (de regulation). Mutations that spontaneously initiate signaling, eg spontaneous Mutations that cause agglutination occur in deregulated growth of tumor cells Plays an important role. The effects of TNF when over-induced are numerous diseases. It is well known that it causes disease. Of intracellular domains (p55ICs) The ability of no p55-TNF receptor to signal independently of TNF is May be contributing to excessive functions such as For example, viruses and Direct cell destruction, with cytopathic effects of other pathogens Not by function but by proteolysis of the intracellular domain of the p55-TNF receptor It may be caused by separation, resulting in TNF-like cytotoxic effects. It appears to be.   Self-association of p55IC and thereby the cause of ligand-independent cytotoxicity Further elucidate the region within p55IC that is the TNF / NGF receptor family Other relevant members (eg, FAS receptors) have the ability to self-associate and To determine whether it has an intracellular domain with ligand-independent action For this purpose, the following detailed research was conducted.(A) General procedures and materials (i) Two hybrid screen and two hive    Lid β-galactosidase expression test    p55-IC and deletion mutants thereof, Fas-IC and various other The cDNA insert encoding the protein (see Table 3) was used in our laboratory. Full-length or cloned cDNA Clones were cloned by PCR from any of the rallies. pGBT-9 and pG AD-GH vector (DNA binding domain (DBD) and activation domain, respectively) Yeast (SFY526) transformed with the cDNA in the main (AD) construct). In a reporter strain (Bartel et al., 1993). β-galactosidase expression in a liquid test (Guarante ), 1983), and further applied to a filter assay. Therefore, the same results were obtained quantitatively (data not shown). p55 receptor Purchased protein for binding to the intracellular domain of the body (p55-IC) HeLa cell cDNA library with Gal4 AD (Clontech , Palo Alto, CA (Ca.), United States) Screening (Fields and Song, 1989) recommended by manufacturer SquatHF7cThis was performed using a yeast reporter strain. Positive of the isolated clone (A) Histidine when grown in the presence of 5 mM 3-aminotriazole The prototrophy of the transformed yeast against ) Due to the expression of β-galactosidase, (c) specific tests (SNF4 and Gal 4 Interaction with lamin fused to DBD).(ii) P55-IC fusion protein produced by bacteria     In vitro self-association   Glutathione S-transferase (GST) and glutathione S- Transferase-p55-IC fusion protein (GST-p55-IC) Other references (Frangioni and Neil, 1993 and Ausubel) bel) et al., 1994). Maltose binding protein (MBP) Transfer the fusion protein to the pMalcRI vector (New England Biolab) (New England Biolabs) Purified on an amylose resin column. Mutual interaction between GST and MBPP fusion proteins Glutathione-agarose beads were continuously fused with GST and MBPP Determined by incubation with protein (5 μg protein / 20 μl beads, first incubation 15 minutes, second 2 hours 4 ° C). Incubation with the MBP fusion protein was performed using 20 mM Tris -HCl, pH 7.5, 100 mM KCl, 2 mM CaCl_Two, 2 mM M gCl_Two5 mM, dithiotreitol, 0.2% triite X100, 0.5 mM phenyl-methyl-sulfonyl-fluoride (phenyl buffer containing 5-methyl-sulphonyl-fluoride) and 5% (v / v) glycerol MgCl in solution or as indicated_TwoInstead of 5 mM EDTA, or Performed in the same buffer containing 0.4M KCl. Association of MBP fusion proteins Is the SDS polyacrylic of the protein associated with glutathione-agarose beads. Luamide gel electrophoresis (10% acrylamide) followed by Western block And evaluated by The blot was treated with a rabbit antiserum against MBP (inventor's Produced in the laboratory) and horseradish peroxidase-conjugated goat anti-rabbit immunity Globulin).(iii) p55 receptor and fragments thereof in HeLa cells      Induced expression   Controlled by tetracycline developed by Gossen and Bayard HeLa cells (HtTA-1 clone (Gossen and Bayard, 1992)) with 10% fetal bovine serum, 100 u / ml penic Phosphorus, 100 μg / ml streptomycin and 0.5 mg / ml neoma Dulbecco's modified Eagle's medium). Insertion of cDNA encoding p55 receptor or part thereof The input was transferred to an expression vector controlled by tetracycline (pUHD10 -3, provided by H. Bujard). Cells By the calcium acid precipitation method (Ausbel et al., 1994) Cells were transfected with the expression construct (5 μg DNA / 6 cm plate) . The effect of transient expression of the transfected protein was As shown after transfection in the presence (1 μg / ml) or absence of Evaluated in between. With the cDNA of human p55-IC in the pUHD 10-3 vector The clones of the stably transfected cells were cDNAs of HtTA-1 cells. Confers resistance to hygromycin in the presence of tetracycline Established by transfection with the plasmid, then Selection of clones resistant to isin (200 μg / ml) was performed. Otherwise, Removal of tetracycline maintained constant in vesicle growth medium increases cDNA expression I got it.(iv) Due to the induced expression of the p55 receptor and fragments thereof     Evaluation of TNF-like action induced   Induced expression of receptors and TNF affects cell viability The effect of neutral Was evaluated by the neutral-red uptake method (Walla ch), 1984). Induction of IL-8 gene expression was assessed by Northern analysis. . RNA is purified using standard procedures using TRI REAGENT (TRI Molecular Research Center (Inc.) And denatured in formaldehyde / formamide buffer. Electrophoresed on a glucose / formaldehyde gel and gel in 10x SSPE buffer. Block on GeneScreen Plus membrane (DuPont) I did it. The filter was used as an IL-8 cDNA probe (Matsushima et al.) 1988, nucleotides 1 to 392). Random-prime kit (Boehringer Mannheim by Okemika (Boehringer Mannheim Biochemica), Mannheim, Federal Republic of Germany Country) and washed rigorously according to the manufacturer's protocol. Oh Autoradiography was performed over 1-2 days.(v) Evaluation of TNF receptor expression   1x10⁶The expression of the TNF receptor in cell samples of Chloras such as Holtmann and Wallach, 1987) Using the chloramine-T method¹²⁵Measurement of binding of TNF labeled with I Was evaluated. The ELISA also lyses the cells (70 μl / 10⁶cel l), RIPA buffer (10 mM Tris) to dilute the sample to be tested -HCl, pH 7.5, 150 mM Na Cl, 1% NP-40, 1%, deoxycholate, 0.1 % SDS and 1 mM EDTA), except that soluble TNF As described for quantification of the condition (Aderka et al., 1991). It was done. A soluble form of the p55 receptor purified from urine was used as a standard.(B) of the p55 receptor to determine the region of p55-IC involved in self-association Mutation analysis of intracellular domain (p55-IC)   As described above, p55-IC can self-associate and exert a cytotoxic effect on cells. P55-I that can be rubbed and further bind to full-length p55-IC There is a C part. In particular, one of the parts of p55-IC (in the first embodiment, (Named protein fragment 55.1) binds strongly to full-length p55-IC And the sequence was further sequenced to give p55-IC Contains amino acid residues 328-426 of the p55-TNF receptor Was done. In addition, said part, the protein fragment 55.1, is itself self-assembled And can cause cytotoxic effects on cells (see below). See). Therefore, this part of p55-IC is called the “dead domain” The region between about amino acid residues 328-426 of the human p55 receptor, most likely Potential is a region consisting of amino acid residues between residues 328 and 414 Exists.   The fact that the "dead domain" in p55-IC self-associates is unexpected Found by the event. this Two-hybrid technology for proteins that bind to the intracellular domain of the receptor Screening of HeLa cell cDNA library by surgery (see Example 1 above) )) Specifically binds to the intracellular domain-GAL4 DBD fusion protein. Among the cDNAs that produce matching products, they are themselves p55 receptor intracellular domains. Some encoding parts of the in (p55-IC, starred in Table 3) Clones (eg, 55.1 and 55.3) were detected.   Assessing the degree of specificity in the self-association of p55-IC and related The application of the two-hybrid test to more accurately define the area will: The findings were derived (Table 3). (A) p55-IC self-association is "dead domain" Within the area. Its N-terminus is located between residues 328 and 344 , Its C-terminus is near residue 404, and the reported C-terminus of this domain (residues 414 ) Is located slightly upstream. (B) of p55-IC upstream of the “dead domain” Deletion of the region close to the membrane enhances self-association, which allows this region to associate Is suggested to have an inhibitory effect on (C) Mouse p55-IC is self And the "killing domain" of the human p55 receptor. (D) TNF / NGF receptor family, three other receptors, namely Fas / APO1 ( FAS receptor), CD40 (Fields and Song, 1989) and p7 Examination of self-association of the intracellular domain of the 5TNF receptor (Smith et al.) Cell death by a sequence motif related to the “killing domain” of the p55 receptor Then sig Nulling Fas-IC must self-associate and to some extent associate with p55-IC It has been shown. However, growth stimulatory signals CD40-IC (e.g., containing sequences similar to the "killing domain") P75-IC, which has no similar structure to p55-IC) It does not self-associate and does not bind to p55-IC or Fas-IC.   Table 3 shows the interaction of the Ga14 hybrid construct including the following proteins. 2 shows a quantitative evaluation for Intracellular domain of human p55 receptor and its various deletions No mutants (numbered in (Loetscher et al., 1990). Residues); intracellular domain of mouse p55 receptor ((Goodwin) Res. 334-454), numbered in Mouse Fas / APO1 ((Watanabe-Fukanaga et al., 1992) Fas-IC, numbered 166-306); human CD40 ((starting CD40-I numbered in Stamenkovic et al., 1989) C, 216-277) and the human p75 TNF receptor ((Smith, 1 990), p75-IC, 287-461). SNFI And SNF4 were used as positive controls for the meeting (Fields ) And Song, 1989), and Ramin as negative controls (Bartel et al., 1993). Gal4 DBD construct ( pGPT9) are listed vertically and encode the Gal4 AD construct (pGAD9). -GH) are listed beside. PGB with the two deletion mutants marked with a star Using p55-IC cloned in T9, HeLa cell cDNA library Cloned in the Library Two Hybrid Screen Clontech, Palo Alto, California, Amelie United States of America). On the screen, about 4 × 10⁶CDNA clone of Four of them were positive. Three of these clones correspond to the cDNA portion of the human p55 receptor. (Two were identical and encoded residues 328-426, one was residue 277 ~ 426). These four can encode unknown proteins all right. The data for β-galactosidase expression was obtained for two independent transformants. The mean of the run-out and the amount of β-galactosidase product (OD of yeast culture)₆₀₀Divided by 10^ThreeDouble OD₄₂₀Activity unit defined as). Analysis detection limit The field was 0.05 units. Deviation between duplicate samples is flat in all cases. Less than 25% of the average (not tested).   Bacterial fusion of p55-IC-glutathione-S-transferase (GST) Protein and p55-IC-maltose binding protein (MBP) fusion protein In vitro studies of interaction with quality confirm p55 receptor self-association However, it was found that yeast proteins were involved in this association (see above). ). The association was not affected by high salt concentration, but by EDTA (see above). See).   To assess the functional involvement of the killing domain in self-association, the p55 receptor, Or the induced expression of that portion affects cells that are sensitive to TNF cytotoxicity The effect was investigated. The result of this analysis is the p55 receptor, its intracellular domain (p 55-IC) or a portion thereof (including the “dead domain”). Independent triggering of cell destruction in HeLa cells ( FIG. 7 showing triggering is described.   FIG. 7 shows the vector transfected with HeLa cells. DNA molecules encoding different types of TNF receptors contained in And the expression vector controlled by tetracycline was used. , Various full-length p55 receptors (p55 receptor), p55-IC or p55-I C part or luciferase (LUC) as a control (first in FIG. 7) Expression in HeLa cells transiently expressing And middle bar graph) and viability (right bar graph) are shown as illustrations. White outline The bar graphs (left, middle and right) show the presence of tetracycline (1 μg / ml). Indicates cells transfected in the presence, said cells inhibiting expression and Bar graphs (left, middle and right) show trans-formation in the absence of tetracycline. Shows the cells that were transfected. TNF receptor expression against the extracellular domain of the receptor ELISA (see the explanatory diagram on the left side of FIG. 7) and radioactivity to cells using antibodies 20 h post-transfection, both by measuring the binding of labeled TNF Later evaluated. The cell-destroying effect of the transfected protein is Evaluation was made 48 hours after the injection. Data shown are quantitatively less Resulting from one of the three experiments, each construct was duplicated. Solid. ND indicates not measured.   Therefore, the transactivator regulated by tetracycline from FIG. Strict control of the transfected cDNA by transactivator By using an expression vector that permits controlled expression (Gossen ) And Bujard, 1 992), of the transiently transfected cDNA for the full-length receptor. Mere increase in p55 receptor expression in HeLa cells by expression is very extensive It is clear that this results in cell death. expressing only p55-IC Greater cytotoxicity was observed than during tobacco. In HeLa cells essentially " Expresses only the part of p55-IC (residues 328-426) consisting of the "dead domain" If so, significant cytotoxicity was also observed. On the other hand, if the "dead domain" Or expression of a part of p55-IC containing only that part (or unrelated control Expression of luciferase, which was used as a I didn't. The cytotoxicity of p55-IC is further enhanced by its stable transformation with its cDNA. Cells that were not induced to express p55-IC. Proliferation continued during tobacco but died when p55-IC was expressed (previously Note).(C) Other actions of the intracellular domain of p55TNF receptor   The other activity of TNF is that of its intracellular domain, including its "dead domain". To determine if it is initiated by an association, (Matsushima et al., 1988), interleukin 8 (IL-8) transcription, increased expression of full-length receptor (p55 receptor) The current effect or the effect of expression of the intracellular domain of the receptor (p55-IC) was examined. . Using constructs controlled by tetracycline, the p55 receptor or Tiger with p55-IC Independent of IL-8 gene ligand in transfected HeLa cells Indicate induction ("General Producers and Materials (Genera l Procedures and Materials) "and Example 1), and the results are shown in FIG. You. FIG. 8 panel A shows RNA extracted from HeLa (HTta-1) cells. (7 μg / lane), untreated cells (control), TNF (500 μ / ml, 4 hours) or p55-IC ("p55-IC"), p55- Receptor ("p55-R") or luciferase ("LUC") cDNA 24 hours after transfection (with or without tracycline) Northern blot analysis of HTta-1 cells after the HTta-1 cell (see “General Process Duplicate of a Northern blot (see Newwards and Materials) reproduction). Each sample shown in panel A of FIG. 8 is included in panel B of FIG. Of Northern blot showing methylene blue staining of 18S rRNA in Show the drafting.   Therefore, as is evident from FIG. 8, the template encoding the p55 receptor cDNA Transfection of HeLa cells with tracyclin-controlled constructs Induction induced IL-8 transcription. Transfection with cDNA of p55-IC A stronger induction was observed in the affected cells. In both cases, Induction occurred only when tetracycline was omitted from the cell growth medium. this thing Expression of p55 receptor or p55-IC transfected with said induction Is shown as a result of Lucifera as control Transfection with thease cDNA had no effect on IL-8 transcription. won.   Thus, from the above results (FIG. 8), a mere increase in p55 receptor expression Or expression of only its intracellular domain (p55-IC), F) In a manner independent of cytotoxicity and expression of IL-8 gene in cells It is clear that it is sufficient to initiate other effects, including the effect of increased is there. Triggering of these actions is triggered by the intracellular domain of the p55 receptor (p55- It is thought to be due to the self-association of IC). As described above, p55-I In the self-association of C, its “dead domain” is first signal of induction of intracellular processes. Triggering intracellular cytotoxicity and other effects Ie, the signaling leading to the induction of IL-8 gene expression is in addition to p55-IC The self-association continues. Therefore, p55-IC The different regions may have different TNF-induced actions (eg, cytotoxicity). , IL-8 induction), and these effects are responsible for p55-IC self- This is the result of intracellular signaling during the association.   p55-IC has other intracellular effects, in a manner independent of ligand (TNF), For example, the fact that it can induce the triggering of IL-8 induction is due to the fact that p55-IC Or a specific portion thereof requires the treatment of cells or tissue to be treated with TNF But rather highly specialized to produce such effects in such cells or tissues. It means that it can be used as a different tool. Many pathological conditions For example, in malignant conditions), treatment with TNF, especially at higher doses, The number of systemically induced intracellular effects by TNF following binding to the NF receptor occurs. Can lead to undesirable side effects. p55-IC is specific to other TNF According to the invention, it can mimic the effect induced (in addition to cytotoxicity). For example, IL-8 induction can be performed in a cell or tissue specific manner. A method for introducing p55-IC or a specific portion thereof has been developed, and a specific desired Signaling to induce intracellular effects, such as induction of IL-8, thereby Will overcome systemic side effects often seen during treatment with TNF .(D) Intracellular domains of TNF receptor and FAS receptor (Fas / APO1) Of cell destruction in HeLa cells by its and "killing domain" Independent triggering   Intracellular domains of p55TNF receptor and FAS receptor (p55IC and With regard to the cytotoxic activity of FAS-IC), p55IC, Extinction domain "(p55DD) and FAS-IC are both present in HeLa cells It has also been elucidated that it can be triggered independently of ligands for cell destruction ing. In this study, HeLa cells were converted to full-length p55-TNF receptor, p55 55IC and portions thereof including p55DD or any of the FAS-ICs Transfected with an expression vector containing the appropriate construct. In one set of experiments, He La cell construct comprising p55TNF receptor (p55-R) and FAS-IC Co-transfected with (See above for details of the construct, its generation, etc.). The results of this study are shown in FIG. 9 (A and Shown in B). Here, in both FIGS. 9A and 9B, HeLa cells were transfected. The construct used to sfect is shown schematically in the left panel; TNF or Shows the results of FAS receptor expression in two middle panels as graphs (2 from the left). And third panels); and the viability of the transfected cells The results are shown graphically in the right panel. In FIG. 9A, in all cases, Using an expression vector controlled by tetracycline, Receptor, p55-IC or a part thereof, or lucifera as a control Of Transfected HeLa Cells Transiently Expressing Lysase (LUC) Is shown. In FIG. 9B, the expression vector controlled by tetracycline To temporarily transfect FAS-IC alone or together with p55 receptor. 2 shows the results for transfected HeLa cells expressed in E. coli. FIG. 9A And in the graphical representation of the results in FIG. 9B, open bars inhibit expression Cells transfected in the presence of tetracycline (1 μg / ml) Vesicles, filled bars transfected in the absence of tetracycline Represents the cells that were performed. TNF receptor expression is an anti-cell against the extracellular domain of the receptor. Body-based ELISA (left panel) and binding of radiolabeled TNF to cells 20 hours post-transfection, by both combined measurements (middle panel) Was evaluated. Transfection of cell destruction effect of transfected cells 48 hours later. Show Data from one of three experiments with quantitatively lower results Each construct was tested in duplicate. The indication “ND” in FIGS. 9A and 9B is Means not. From the results shown in FIGS. 9A and 9B, only p55IC It is clear that the current results in more cytotoxicity. Its extinct Significant cytotoxicity also occurs when only the main (p55DD) is expressed. Contrast Thus, expression of a portion of p55IC that lacks or contains only a killing domain is It had no effect on cell viability. Expression of FAS-IC resulted in Does not result in significant cytotoxicity, only the cytotoxicity of the co-expressed p55 receptor It was only significantly increased.Example 3 Additional tags capable of binding to the intracellular domain of the p55TNF receptor or the FAS receptor Protein   Using similar approaches and techniques as described in Example 1 above, an additional 3 Proteins capable of binding to two p55IC or FAS-IC were isolated and identified.   In FIGS. 10-12, cDNAs referred to as F2, F9 and DD11, respectively. Schematic representation of clone parts and preliminary nucleotide sequences You.   Mouse embryo using mouse FAS-IC with clones F2 and F9 as "feed" The library was isolated by screening. FIG. The partial nucleotide sequence derived from the F2 cDNA is shown schematically. In FIG. Partial nucleotide sequence of 1724 bases from sequenced F9 cDNA Diagrammatically You.   The proteins encoded by clones F2 and F9 (F2 and F9, respectively) And the binding capacity of F9) (A) F2 with human p55IC and p55DD, and mouse FAS-IC Strongly interacts with, but not as suitable as, suitable for human FAS-IC Interacts only weakly with the (control) proteins SNFI and Lamin What not to do (B) F9 interacts strongly with human p55-IC and mouse FAS-IC Is human FAS-IC (suitable protein) and inappropriate protein SNFI And interacts only weakly with Ramin, (C) Both F2 and F9 are human p75IC, pGBT9 (empty bait vector), human CD Did not interact with -40 at all Was shown.   Furthermore, when searching "gene bank" and "protein bank", And F9 were found to represent a novel protein.   Thus, F2 and F9 bind to both FAS-IC and p55IC It means a novel protein with specificity.   Human HeLa clone using mouse p55DD with clone DD11 as “feed” Isolated by screening the libraries. In FIG. 12, the sequence was determined. Schematic representation of the partial nucleotide sequence of 425 bases from DD11 cDNA. You.   The DD11 clone has a length of about 800 nucleotides. Total length of transcript is about 1 2 kb and probed with the clone. By clone DD11 Analysis of the binding capacity of the encoded protein indicated that DD11 was p55DD ( Amino acids 326-414) (see FIG. 9), but this domain Does not interact with deletion mutants, e.g., amino acids 326-404. DD1 1 also interacts with mouse and human FAS-IC, and to some extent Interacts with DD11 is both SNF1 and pGBT9 (empty bait vector) No interaction at all. DD11 is also a “gene bank” and “protein” It was not in the Bank's database. Therefore, DD11 is p55IC (p 55DD) and FAS-IC specific binding proteins.Example 4 Construction of soluble dimeric TNF receptor   Based on the results of the study described in Example 2, the intracellular domain of the p55 receptor (P55-IC) and portions thereof (the "dead domain"), and Fas / APO1 and its portion analogous to the p55-IC "dead domain" (" Killing domain) can self-associate, self-associate (aggregate) and Certain new TNF receptors can be constructed. Such a TNF receptor is p 55 receptor or the intracellular domain of Fas / APO1 or its “dead domain” Has all of the extracellular domain of the p55 receptor fused to essentially all of Become a fusion protein. Therefore, such fusion The combined construct is a transmembrane domain of the p55 receptor (or FAS / APO1). It will lack mains and is therefore soluble. In addition, its intracellular domain By virtue of the self-association ability of the killing or “dead domain”, these fusion constructs At least a dimer of the p55 receptor (and potentially a larger order) Multimers) also allow for the provided oligomerization. Then, such a da The imer TNF receptor (p55 receptor) is one of the few naturally occurring TNF homotrimers. Can bind to at least two TNF monomers and their ligands (TNF homotrimers) To provide a soluble TNF receptor that binds more avidly.   Thus, at least four types of p55TNF receptor fusion proteins are constructed Each of which can be oligomerized and soluble,   (i) p55 receptor extracellular domain (EC55) and p55 receptor intracellular domain Fusion product with in (p55-IC),   (ii) Fusion product of EC55 and “dead domain” of p55-IC (DD55) ,   (iii) Fusion of EC55 with the intracellular domain of Fas / APO1 (IC FAS) Product and   (iv) Fusion of EC55 and IC FAS “dead domain” (DD FAS) Adult It is.   In each of the fusion proteins, the TNF monomer binding capacity was EC55 Although provided by the moieties, oligomerization (or less) of each type of fusion protein Without Is also a dimer) is the p55IC, DD55, IC FAS or DD FAS portion Is provided by its "tail" region.   For the construction of the fusion protein, standard techniques of recombinant DNA technology are used. Technique can be used, which is now well established in the art (See, for example, Sambrook et al., (1989), Molecular Clos. Cloning: A Laboratory Manual (A Laborato) ry Manual), Cold Spring Harbor Laboratory Press (Cold) Spring Harbor Laboratory Press), Cold Spring Harbor, New York)). Briefly, of any suitable bacteria, bacteriophages Or an animal virus expression vector (designed for expression of the selected insert DNA) Cloned vehicles or plasmids) can also be used, in which EC55 and p55-IC, DD55, IC FAS or DD FAS DNA encoding one of the "tails" is inserted in one or more steps. Said Such inserted DNA encoding each of the fusion proteins may be Cloning of promoters, ribozyme binding sites, transcription factor binding sites, etc. It is under the control of various expression control sequences of the vehicle or plasmid. this Such expression control sequences will depend on the type of Of a host cell (eukaryotic or prokaryotic) in which it is desired to express a fusion protein of The choice depends on the type. Preferred host cells (and therefore expression vectors) Is a eukaryotic cell And particularly preferably mammalian cells.   DNA molecules encoding each of the above fusion proteins are obtained by the following procedure. Prepared and inserted into expression vector. (A) First, a set of oligonucleotides for use in PCR is prepared by standard means, Constructed with oligonucleotides that are (B) Full-length cloned p55 receptor and F in our laboratory Plasmid containing as / APO1 receptor (co-pending EP56) 8925 and Examples 1-3) were subjected to the following procedures to DNA encoding each of the proteins A fragment is generated, and the DNA fragment is then ligated into the selected expression vector.   (i) D encoding EC55, a component of all four fusion proteins To prepare an NA fragment, use the oligonucleotides Nos. 1 and 2 (fragment size) Plasmid carrying the human p55 cDNA using Perform R.   (ii) Oligonucleotide number 3 to obtain the fusion product EC55-IC55 PCR was carried out on plasmids carrying the human p55 cDNA using A DNA fragment coding for IC55 (size 677 bp) was obtained. Mix with SalI cut EC55 and place under control of a suitable promoter. The expression vector for mammalian cells is ligated with a smooth end ligation. To the vector Orientation of the inserted EC55-IC55 is restricted by restriction digestion. And by sequencing.   (iii) To obtain the EC55-IC FAS fusion product, Using Rigonucleotide Nos. 5 and 6 to a plasmid carrying FAS cDNA A fragment (size 448 bp) was obtained by PCR, followed by AllI cut, mixed with SalI cut EC55, followed by the appropriate promoter Under mammalian control by blunt ligation. Before The orientation of the inserted EC55-IC FAS in this vector is Confirm by restriction digest and by sequencing.   (iv) To obtain the fusion product EC55-DD55, DN The A fragment was converted to human p55 cDNA using oligonucleotides 7 and 4. And using the DD55 sequence by PCR. Generation of size 314bp The product was cut with SalI, mixed with SalI cut EC55 and then smoothed. The ligation ligates to a mammalian expression vector. Inserted in the vector The orientation of EC55-DD55 was determined by restriction digestion and by sequencing. Confirm.   (v) To obtain the fusion product EC55-DD FAS, The NA fragment was ligated to the FAS cDNA using oligonucleotides 6 and 8. Prepared by PCR. The 332 bp product was cut with SalI Mixed with EC55 cut with SalI, followed by blunt ligation in mammalian expression Connect with vector. Next, the EC55-DD FAS orientation was restricted. Confirm by restriction digestion and sequencing.   Once the expression vectors have been constructed, they can then be used for expression purposes. Suitable mammalian cells (for example, Chinese hamster ovary (CHO) cells or Or monkey kidney (COS) cells) by standard methods. Expressed in that way The fused fusion protein was then prepared in a standard manner (co-pending EP 308378). , EP 398327 and EP 568925). Purified fusion The proteins then have the ability to oligomerize (and to what extent Whether to form dimers or larger multimers) and TN The ability to bind F (and the affinity or aggressiveness of that binding) Analyze.Example 5 Construction of soluble dimer Fas / APO1 receptor   In the same manner as described in Example 4, the following four types of Fas / APO1 fusions The product can be made. Each of them can be oligomerized, Is soluble,   (i) Extracellular domain of Fas / APO1 (EC FAS) and p55-IC Fusion products with intracellular domains,   (ii) Fusion of EC FAS and p55-IC “dead domain” (DD55) Adult,   (iii) EC FAS and intracellular domain of Fas / APO1 (IC FAS) Fusion products and   (iv) Integration of EC FAS and IC FAS “dead domain” (DD FAS) Synthetic product It is.   For each of the fusion proteins, the FAS ligand binding capacity was EC F As provided by the AS moiety, each type of oligomerization of the fusion protein (also Is at least dimerization) is p55-IC, DD55, IC FAS or DD   Provided by its "tail" area, which is one of the FAS parts.   DNA fragment encoding the fusion protein and expression vector containing them Constructs different suitable oligonucleotides (not shown) with the "tail" region described above. Example 4 except that it is used to prepare EC FAS fragments that are ligated to any of the regions. It is described in detail. Subsequently, the expression vector is introduced into a suitable host cell, and the results and The resulting expressed fusion protein is purified and oligomerized. Their ability (and to what extent, that is, whether they are dimers or larger Whether to form multimers) and the ability to bind FAS ligands (And its binding affinity or aggressiveness).Example 6 Construction of soluble oligomer "mixed" TNF / FAS receptors   "Mixed" affinity, ie both TNF and FAS receptor ligands To prepare an oligomeric receptor having affinity for The fusion products of Examples 4 and 5) can be used in the following procedure. i) any one of p55IC, FAS-IC, p55DD or FASDD Of TNF receptor (p75TNF receptor or p55TNF receptor) fused with Providing a fusion product as described in Example 4 comprising an extracellular domain; ii) Any one of p55IC, FAS-IC, p55DD or FAS-DD Fusion production as described in Example 5, comprising the extracellular domain of the Fas receptor fused to Providing things, iii) combining any one of the fusion products of i) with any one of the fusion products of ii) Mixed, having both the extracellular domains of the TNF and FAS receptors, Novel dimers (or better) where they are linked by their IC or DD regions To provide larger order oligomers) receptors.   In the above procedure, the fusion proteins of i) and ii) are provided separately, That is, to produce the protein Provided from their purified product from transformed cells and then mixed in vitro A mixed affinity receptor may be obtained. Alternatively, host cells can be transformed with both types of fusion It may be co-transfected with a vector carrying the sequence encoding the protein, In some cases, the mixed affinity receptor is a co-transfected cell May be obtained directly from The actual oligomerization of the fusion protein into oligomers is detailed. During purification procedures to obtain fusion products expressed intracellularly or in cells May be performed after the above procedure. Select Mixed Affinity Receptors Any standard method may be used for this, for example, TNF receptor and Chromatography of antibodies to the extracellular domains of the FAS and FAS receptors To select those receptors with both types of extracellular domains.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI C07H 21/04 9356-4H C07K 14/47 C07K 14/47 9735-4B C12N 1/19 C12N 1/19 9735- 4B 1/21 1/21 9637-4B C12P 21/02 C 5/10 7823-4B C12Q 1/68 A C12P 21/02 0276-2J G01N 33/566 C12Q 1/68 9356-4H C07K 14/48 G01N 33 / 566 9356-4H 14/525 // C07K 14/48 9735-4B C12N 5/00 B 14/525 9051-4C A61K 37/02 (C12P 21/02 C12R 1:19) (C12P 21/02 C12R 1: 645) (C12P 21/02 C12R 1:91) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, B J, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, LU , LV, MD, MG, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, UG, US, UZ, VN (72) Inventor Met, Igoa, Israel, 76462 Rehoboth, Levin Epstein Street 60 (72) Inventor Balforomef, Eugene Israel, 76100 Rehoboth, Pio Box 26, Wiseman Institute of Science, Department of Membrane Research and Biophysics (None)

Claims (1)

[Claims] 1. Tumor necrosis factor / nerve growth factor (TNF / NGF) receptor superfamily A receptor capable of binding to one or more intracellular domains of one or more receptors belonging to DNA sequence encoding a protein. 2. The receptor is a TNF receptor, a p55TNF receptor or a p75TNF receptor Or a FAS ligand receptor (FAS receptor). DNA sequence of 3. (a) Intracellular domain binding protein of natural TNF receptor or FAS receptor A cDNA sequence from the region encoding the quality,   (b) hybridizing with the sequence of (a) under moderately stringent conditions. , And biologically active intracellular domain binding of TNF or FAS receptors A DNA sequence encoding a protein, and   (c) as a result of the genetic code, degenerate with the DNA sequence defined in (a) and (b), And biologically active intracellular domain binding of TNF receptor or FAS receptor DNA sequence encoding protein   The DNA sequence according to claim 1 or 2, which is selected from the group consisting of: 4. Coding of p55TNF receptor intracellular domain (p55IC) binding protein 4. The DNA sequence according to claim 1, 2 or 3, wherein the DNA sequence is modified. 5. Named protein, 55. 1, 55. 3, 55. 11, F2, F9 and And DD11 The DNA sequence according to claim 4, which encodes a protein to be produced. 6. Designated cDNA clones, 55. 1, 55. 3, 55. 11, F2, F The DN according to claim 5, wherein the DN is selected from the sequences included in DD9 and DD11. A sequence. 7. Coding of the p75TNF receptor intracellular domain (p75IC) binding protein 4. The DNA sequence according to claim 1, 2 or 3, wherein the DNA sequence is modified. 8. Named protein, 75. 3 and 75. Selected from the group of 16 The DNA sequence according to claim 7, which encodes a protein. 9. Designated cDNA clones, 75. 3 and 75. Sequence included in 16 The DNA sequence according to claim 8, which is selected from the group consisting of: Ten. Amino acid residues 328 to 426 of the amino acid sequence of the p55TNF receptor 55. Protein having a amino acid sequence Claim 5 or Claim 1 which codes 1 7. The DNA sequence according to claim 6. 11． Amino acid residues 277 to 426 of the amino acid sequence of the p55TNF receptor 55. Protein having a amino acid sequence Claim 5 or Claim 5 which codes 3 7. The DNA sequence according to claim 6. 12． 55. Protein consisting of the sequence shown in FIG. Code 11, bill 7. The DNA sequence according to item 5 or 6. 13. A protein 75 consisting of the sequence shown in FIG. Code 3 Item 8 or 9 DNA sequence. 14. A protein 75 consisting of the sequence shown in FIG. Code 16, Request 10. The DNA sequence according to item 8 or 9. 15． Encodes a FAS receptor intracellular domain (FAS-IC) binding protein 4. The DNA sequence according to claim 1, 2 or 3. 16． Selected from the group consisting of the named proteins, F2, F9 and DD11 16. The DNA sequence according to claim 15, which encodes a protein. 17． The sequence contained in the named cDNA clone, F2, F9 and DD11 17. The DNA sequence according to claim 16, which is selected from the group consisting of: 18． Encodes any of the proteins, F2, F9 and DD11; Claim 16 or Claim 17 comprising the arrangement shown in any of Figures 10-12. 18. The DNA sequence according to claim 17. 19. One or more intracellular doses of one or more TNF receptors or FAS receptors An arrangement according to any one of claims 1 to 18, which can be coupled to the main. The protein encoded by the sequence, or analogs and derivatives thereof. 20. Protein, 55. 1, 55. 3, 55. 11, 75. 3, 75. 16, F 20. The tag according to claim 19, which is selected from the group consisting of 2, F9 and DD11. Proteins, and biologically active analogs and derivatives. twenty one. Having the deduced amino acid sequence shown in FIG. The protein according to claim 20, 55. 11. twenty two. p55-TNF in the region of residues 243-308 of the p55-TNF receptor Further characterized by being able to bind to the intracellular domain of the receptor (p55IC) 22. The protein of claim 21, which is isolated. 11. twenty three. A DNA sequence according to any one of claims 1 to 18. vector. twenty four. 24. The vector according to claim 23, which can be expressed in a eukaryotic host cell. - twenty five. 24. The vector according to claim 23, which can be expressed in a prokaryotic host cell. - 26. A form comprising the vector according to any one of claims 23 to 25. Transformed eukaryotic or prokaryotic host cells. 27. 21. A protein, analog or derivative according to claim 19 or 20. The method according to claim 26, wherein the transformed host cell according to claim 26 is Growing under conditions suitable for the expression of the protein, analog or derivative; Performing post-translational modification of the protein as necessary to obtain the protein; Expressing the expressed protein, analog or derivative from the culture of the transformed cells or A production method comprising extracting the transformed cell from a cell extract. 28. 21. A protein, analog or derivative according to claim 19 or 20. Or an active fragment or derivative thereof. 29. TNF to cells bearing TNF or FAS receptors 20. A method for modulating the action of NF or FAS receptor ligand, wherein the method comprises the steps of: Or the proteins, analogues and derivatives of paragraph 20 and p55IC, p 55DD, FAS-IC or FAS-DD, an analogue or derivative thereof. All the proteins are intracellular domains of the TNF receptor or FAS receptor That can modulate TNF receptor or FAS receptor activity by binding to One or more proteins, analogs and proteins selected from the group consisting of Treating the cell with a conductor, wherein the treatment of the cell comprises the one or more cells. Introducing the protein, analog or derivative into the cell in a form suitable for introduction into the cell. Encoding the one or more proteins, analogs or derivatives From introducing the DNA sequence into the cell in the form of a suitable vector carrying the sequence The vector inserts the sequence into the cell such that the sequence is expressed in the cell. Adjustment methods that can bring. 30. The treatment of the cells is performed by transducing the cells with a recombinant animal virus vector. Due to the fection,   (a) Specific cell surface on the surface of cells bearing TNF receptor or FAS receptor A sequence encoding a viral surface protein (ligand) capable of binding to the receptor; 21. The protein, analog or derivative according to claim 19 or 20. Rabi ni p55IC, p55DD, FAS-IC or FAS-DD, analogs thereof Or when the protein is expressed in the cell. A protein capable of regulating the activity of the TNF receptor or the FAS receptor A recombinant animal carrying a second sequence encoding a protein selected from the group consisting of: Constructing a viral vector, and   (b) a step of infecting the cells with the vector of (a)   30. The adjusting method according to claim 29, comprising: 31. TNF or FAS receptor for cells bearing TNF or FAS receptor A method for regulating the action of a ligand, comprising the antibody or the activity thereof according to claim 28. Treating the cells with a sexual fragment or derivative, wherein the treatment is directed against the cells. Of a suitable composition containing the antibody or an active fragment or derivative thereof When the IC binding protein of the cell is exposed to the extracellular surface, The composition is formulated for extracellular application, wherein the IC binding protein is administered intracellularly. Wherein the composition is formulated for intracellular application. 32. TNF or FAS receptor for cells bearing TNF or FAS receptor A method for regulating the action of a ligand, wherein the method comprises any one of claims 1 to 18. A sequence encoding an antisense sequence of at least a portion of the sequences described in And antisense sequences of p55IC, p55DD, FAS-IC or FAS-DD Treating the cells with an oligonucleotide sequence selected from the sequence encoding the sequence The oligonucleotide sequence of the TNF receptor or the FAS receptor Able to block expression of at least one of the intracellular domain binding proteins Adjustment method. 33. The oligonucleotide sequence is the second sequence of the virus is the oligonucleotide 31. The cell according to claim 30, which encodes a peptide sequence. 33. The adjusting method according to claim 32, wherein the adjusting method is performed. 34. A method of treating tumor cells or HIV infected cells or other pathological cells. hand,   (a) a specific tumor cell surface receptor or HIV infected cell surface receptor or Virus surface proteins that can bind to receptors carried by other pathological cells 21. A sequence encoding a protein, and a protein according to claim 19 or 20. , Analogs and derivatives and the p55TNF receptor intracellular domain (p55IC ), Its “dead domain” (p55DD), the intracellular domain of the FAS receptor (F AS-IC) or its “dead domain” (FAS-DD), or biologically An active analog or derivative thereof, wherein said tumor cell, said HIV infected cell, or A protein capable of killing said other pathological cells when expressed in said cells Recombinant animal virus carrying a sequence encoding a protein selected from the group consisting of: Constructing a lus vector, and   (b) the tumor cell or HIV-infected cell or other pathological cell is treated with the vector of (a); The process of infecting   A treatment method consisting of: 35. A method for inducing a TNF-related effect in a cell or tissue, comprising essentially all Is the self-associating intracellular domain of the p55TNF receptor (p55-IC) or Selected from proteins that are part of it that can be associated, Treating the cells with one or more proteins, analogs or derivatives thereof And inducing the TNF action in a cell independently of the ligand (TNF) The treatment of the cell comprises administering to the cell the one or more proteins, analogs or Introducing the derivative in a form suitable for introduction into the cell, or introducing the derivative into the cell. Alternatively, a DNA sequence encoding a plurality of proteins, analogs or derivatives In the form of a suitable vector that carries the sequence. Induction method capable of effecting insertion of said sequence into said cell to be expressed in the cell . 36. The treatment of the cells transfects the cells with a recombinant animal virus vector Done by   (a) Will capable of binding to a specific cell surface receptor on the surface of the cell to be treated A sequence encoding a surface protein (ligand); P55 capable of self-associating and inducing the one or more TNF-related effects -Coding of ICs, parts thereof, proteins which are analogs and derivatives of all of the foregoing. Constructing a recombinant animal virus vector carrying a second sequence to be loaded; and   (b) infecting the cell with the vector of (a)   36. The guidance method according to claim 35, comprising: 37. The TNF action induced in the cells is induction of IL-8 gene expression; The vector comprises essentially all of the p55-IC, a portion thereof, and all of the foregoing. Carrying the sequence encoding all analogs and derivatives, and the protein is Self-meeting when revealed, said Claims that can perform signaling that induces expression of the IL-8 gene. Item 35. The guidance method according to Item 35 or 36. 38. Antibacterial for treating tumor cells or virus-infected cells, or for granulocytes The virus vector for enhancing the effect is the tumor cell, the virus-infected cell, Or a viral ligand capable of binding to a specific cell surface receptor on the surface of granulocytes Encoding sequence, and said p55-IC, parts thereof, analogs and derivatives thereof And the protein is the tumor cell, the virus-infected cell Or TNF-related work that, when expressed in granulocyte cells, results in the death of these cells. The guidance method according to any one of claims 35 to 37, wherein the guidance is performed. Law. 39. A method of treating a tumor cell, said p55-IC, a part thereof, an analogue thereof Or when the derivative is expressed in tumor cells, it can cause granulocytes and other lymphocytes to The IL-8 chemotactic activity attracts cells to kill tumor cells 39. The inducer of claim 38, wherein the inducer induces expression of IL-8, which leads to tumor cell death. Guidance method. 40. For treating cells by inducing TNF-related effects in the cells The intracellular domain of the p55 receptor (p55-IC), portions thereof, Analogues and derivatives. 41. To treat cells by inducing IL-8 gene expression in the cells 41. A p55-IC, moiety, analog and derivative according to claim 40 for use. 42. Inducing IL-8 gene expression in tumor cells resulting in tumor cell killing 42. The p55-I according to claim 41, which is used for treating a tumor cell. C, moieties, analogs and derivatives. 43. A method for regulating TNF or FAS receptor ligand action on cells, comprising the steps of: 21. A cellular mRNA encoding the protein according to claim 19 or 20. Column, or p55IC, p55DD, FAS-IC or FAS-DD Encodes a ribozyme sequence that can interact with the mRNA sequence To the cell in such a form that the ribozyme sequence is expressed in the cell. And the ribozyme sequence is compatible with the cellular mRNA sequence when expressed in the cell. Interact and cleave the mRNA sequence to cleave the protein or the p55IC, p55 Resulting in inhibition of the expression of DD, FAS-IC or FAS-DD in the cell, A regulation method comprising applying a ribozyme method. 44. An intracellular domain-binding protein according to claim 19 or 20, A method for isolating and identifying a protein, factor or receptor capable of binding, comprising: 21. The protein according to item 19 or 20, wherein the protein is affinity chromatography. Binding to a laffy substrate, contacting the bound protein with a cell extract, Next, the proteins, factors and the like derived from the cell Or affinity chromatography methods to elute, isolate, and analyze A method consisting of applying. 45. An intracellular cell according to claim 19 or claim 20. A method for isolating and identifying a protein capable of binding to a main binding protein, A sequence encoding the intracellular domain-binding protein in a first hybrid vector Sequence from the cDNA or genomic DNA library Hybrid vectors, and then use these vectors to transform yeast host cells. Transformed, isolating positive transformed cells, followed by the second hybrid vector. And encodes a protein that binds to the intracellular domain binding protein. A method comprising obtaining the sequence of interest and applying the yeast two-hybrid method. 46. Modulates TNF-receptor or FAS-receptor ligand action on cells; 21. The protein or protein according to claim 19 or 20 p55IC, p55DD, FAS-IC or FAS-DD, Pharmaceutical compositions comprising active fragments, analogs, derivatives or mixtures thereof. 47. Modulates TNF or FAS receptor ligand action on cells, and as an active ingredient Encodes a protein capable of binding to a cell surface receptor, Is the protein according to item 20, or the protein p55IC, p55DD, FA Encodes S-IC or FAS-DD, biologically active fragment or analog thereof A pharmaceutical composition comprising a recombinant animal virus vector. 48. Modulates TNF or FAS receptor ligand action on cells, and as an active ingredient An antisense sequence of the sequence according to any one of claims 1 to 18. Pharmaceutical group consisting of oligonucleotide sequences encoding sequences Adult. 49. Protein that can bind to the intracellular domain of TNF receptor or FAS receptor A method for isolating and identifying a sequence, comprising the steps of claim 1 to claim 18; Uses that part as a probe and has at least partial homology to it, capturing sequences from a cDNA or genomic DNA library, Claims 1 to 18 wherein the sequence is then amplified by PCR and cloned. Encodes a protein having at least partial homology to the described sequences Non-stringent Southern hybridizations and clones A method comprising applying a PCR cloning method followed by 50. Soluble, oligomer comprising at least two self-associated fusion proteins -Tumor necrosis factor receptor (TNF receptor), each fusion protein (A) at one end thereof an extracellular domain of TNF, an analog or derivative thereof; Having a TNF binding domain selected from the group consisting of: Derivatives are harmful substances that prevent TNF binding or reduce optimal TNF binding. Cannot self-associate and bind to TNF; and (b) At the other end, (i) the amino acid of the natural p55TNF receptor molecule (p55 receptor) A p55 TNF receptor that extends from about 206 amino acid residues to about 426 amino acid residues. Essentially all of the intracellular domain (p55-IC); (ii) the native p55 receptor Killed p55-IC extending from about 328 amino acid residues to about 426 amino acid residues Main; (iii) Essentially all of the intracellular domain of the Fas / APO1 receptor (Fas-IC); (iv) the death domain of Fas-IC; and (v) capable of self-association (i)-(i v) a self-associating domain selected from any one analog, fraction or derivative of The at least two self-associated proteins are self-assembled only at the end (b). Each having an end (a) capable of associating with and binding to at least two TNF monomers. End (a) is capable of binding one TNF monomer, a TNF receptor, and The soluble, oligomeric TNF receptor salt or functional derivative. 51. Essentially all native p55 receptors as its at least two termini (a) P55TNF receptor, extending from about 1 amino acid residue to about 172 amino acid residues of The extracellular domain of the body (p55 receptor) and at least two ends (b) thereof 51. The soluble of claim 50, comprising essentially all of said p55-IC. , Oligomeric TNF receptor. 52. Essentially all native p55 receptors as its at least two termini (a) P55TNF receptor, extending from about 1 amino acid residue to about 172 amino acid residues of Essentially all of the extracellular domain of the body and its at least two termini (b) The soluble of claim 50, which comprises the killing domain of the p55-IC of claim 50. Oligomeric TNF receptor. 53. The extracellular domain of the p55TNF receptor as at least its two ends (a) And each of said analogs or derivatives comprises one TNF module. Can bind to the nomer, cannot self-associate, And essentially all of the p55-IC as at least two ends (b) thereof 51. The soluble, oligomeric TNF receptor of claim 50. 54. As its at least two ends (a), the class of the extracellular domain of the p55 receptor Consisting of analogs or derivatives, each of said analogs or derivatives being one TNF monomer And can not self-associate, and at least two of Claim consisting essentially of all said p55-IC killing domains as one end (b) 51. The soluble, oligomeric TNF receptor of claim 50. 55. Essentially all native p55 receptors as its at least two termini (a) P55TNF receptor, extending from about 1 amino acid residue to about 172 amino acid residues of As the extracellular domain of the body and at least its two termini (b) and at least Claims consisting essentially of all said Fas-IC as at least two ends (b) 51. The soluble, oligomeric TNF receptor of claim 50. 56. Essentially all native p55 receptors as its at least two termini (a) Extending from about amino acid residue 1 to about amino acid residue 172 of the p55 receptor As extracellular domain and at least two ends thereof and at least two Consists essentially of the dead domain of all the Fas-IC as one end (b) 51. The soluble, oligomeric TNF receptor of claim 50. 57. As its at least two ends (a), the class of the extracellular domain of the p55 receptor Consisting of analogs or derivatives, each of said analogs or derivatives being one TNF monomer And Can bind, cannot self-associate, and at least 50. The method of claim 50, wherein the terminal (b) consists essentially of all said Fas-IC. Soluble, oligomeric TNF receptor as described. 58. As its at least two ends (a), the class of the extracellular domain of the p55 receptor Consisting of analogs or derivatives, each of said analogs or derivatives being one TNF monomer And can not self-associate, and at least two of Claim 50. Consisting essentially of all said Fas-ICs as one end (b) A soluble, oligomeric TNF receptor according to claim 7. 59. A soluble oligomer according to any one of claims 50 to 58. A method for producing a TNF receptor,   (a) construction of an expression vector encoding any one of the fusion proteins, The DNA sequence at each of the ends of the combined protein is essentially all of the TNF receptor , A cloned D encoding the cell or its domain of an analog or derivative thereof From the NA sequence and essentially all of the p55-IC, p55-IC killed domains In, Fas-IC, Fas-IC killing domain, all analogs of the foregoing Or a cloned DNA sequence encoding a derivative, the ends of which are shared. To form a fusion protein sequence, wherein the fusion protein sequence is And inserted into the vector under the control of translation control sequences,   (b) inserting the vector into an appropriate host cell in which the fusion protein is expressed; and And   (c) before, during, or after a purification process that yields a soluble, oligomeric TNF receptor Fusion protein expressed in the host cell, wherein the fusion protein is self-associated Purification   Manufacturing method consisting of: 60. The fusion protein according to any one of claims 50 to 58, An expression vector comprising a fusion protein sequence to be loaded. 61. Claim 60. A claim 60 for use in the method of claim 59. On the vector. 62. The vector according to claim 60, which is capable of expressing said fusion protein sequence. Host cells. 63. Claims 50 to 50 as pharmaceutically acceptable carriers and active ingredients 59. The soluble, oligomeric TNF receptor according to any one of paragraph 58, a salt thereof or Is a pharmaceutical composition comprising a functional derivative and a mixture of any of the foregoing. 64. Used in antagonizing the deleterious effects of TNF in mammals, especially in excess Used in the treatment of conditions where TNF is formed endogenously or administered exogenously 60. The soluble, oligomer according to any one of claims 50 to 58, wherein A TNF receptor, a salt or a functional derivative thereof, and a mixture of any of the foregoing. 65. Prolongation of TNF in mammals when used with exogenously administered TNF Claims 50 to 58 used in maintaining the beneficial effect obtained. A soluble, oligomeric TNF receptor, salt or functional derivative thereof according to any one of the above. And mixtures of any of the foregoing. 66. Soluble, oligomer comprising at least two self-associated fusion proteins -Fas / APO1 receptor (Fas receptor), each fusion protein The quality can be (a) unable to self-associate to one end and bind to the Fas ligand. Selected from the extracellular domain of the Fas receptor, an analog or derivative thereof. It has a Fas ligand binding domain and (b) at its other end (i) essentially all About 206 amino acid residues of any native p55TNF receptor molecule (p55 receptor) Extending to about amino acid residue 426, the intracellular domain of the p55TNF receptor (P55-IC), (ii) amino acids from about amino acid residue 328 of the natural p55 receptor A killing domain of p55-IC spanning about 426 acid residues, (iii) essentially all All intracellular domains of Fas / APO1 receptor (Fas-IC), (iv) Fas -The killing domain of the IC and (v) similar to any one of (i) to (iv) capable of self-association A self-associating domain selected from a body or derivative; Self-assembled proteins only self-associate at the terminus (b) and have at least two The ends (a) capable of binding to a Fas ligand monomer, each end (a) having one Fa a Fas receptor capable of binding to an s-ligand monomer; Salts or functional derivatives of oligomeric Fas receptors. 67. A method for producing a soluble, oligomeric Fas receptor according to claim 66. So,   (a) construction of an expression vector encoding any one of the fusion proteins, The DNA sequence at each of the ends of the combined protein is essentially all Fas receptors. , That Cloned DN encoding the extracellular domain of an analog or derivative of A sequence and essentially all of the p55-IC, p55-IC killed domains , Fas-IC, Fas-IC killing domain, all of the foregoing Obtained from a cloned DNA sequence encoding an analog or derivative; The ends are joined together to form a fusion protein sequence, wherein the fusion protein sequence is Inserted into the vector under the control of transcription and translation regulatory sequences,   (b) inserting the vector into an appropriate host cell in which the fusion protein is expressed; and And   (c) before, during, or after a purification process that yields a soluble, oligomeric Fas receptor Fusion protein expressed in the host cell, wherein the fusion protein is self-associated Purification   Manufacturing method consisting of: 68. A fusion protein encoding the fusion protein according to claim 66. Expression vector consisting of rows. 69. The vector according to claim 68 for use in the method according to claim 67. . 70. The vector according to claim 68, wherein said vector can express said fusion protein sequence. Host cells. 71. Claim 66. The pharmaceutically acceptable carrier and the active ingredient according to claim 66. Soluble, oligomeric Fas receptor, salt or functional derivative thereof and any of the foregoing A pharmaceutical composition comprising a mixture of the above. 72. For use in antagonizing the deleterious effects of Fas ligand in mammals, particularly Treatment of conditions where excess Fas is formed endogenously or is administered exogenously To 67. A soluble, oligomeric Fas receptor according to claim 66 for use therein. Salts or functional derivatives and mixtures of any of the foregoing. 73. Soluble with affinity for both TNF and FAS receptor ligands, Oligomeric receptor, at least two self-associated fusion proteins Wherein one of the fusion proteins is as defined in claims 50 to 58. A protein derived from a TNF receptor specific to TNF according to any one of the above, 67. The other fusion protein is specific for a Fas receptor ligand of claim 66. A receptor derived from a novel Fas receptor. 74. A pharmaceutical composition comprising the mixed affinity receptor according to claim 73. 75. In antagonizing the deleterious effects of TNF and Fas ligand in mammals 74. A mixed affinity receptor according to claim 73 for use. 76. Pharmaceutical compositions for treating cells by inducing TNF-related effects in the cells And p55-IC, a pharmaceutically acceptable carrier and an active ingredient. 47. The method of claim 46, wherein the moiety comprises all analogs and derivatives of the above. Pharmaceutical composition. 77. Pharmaceutical compositions for treating cells by inducing TNF-related effects in the cells And p55-IC as an active ingredient, a part thereof, all the above-mentioned analogs and the like. Recombinant animal virus vectors encoding the 57. The method according to claim 57, comprising a protein capable of binding to a vesicle surface protein. Medicine Composition. 78. Induction of IL-8 expression and subsequent tumor cell death by administration of the composition 78. The method according to claim 76 or 77 for treating tumor cells, which leads to destruction. Pharmaceutical composition.