CN1616661A - Modulators of TNF/NGF superfamily receptors and soluble oligomeric TNF/NGF superfamily receptors - Google Patents

Abstract

The present invention generally concerns novel proteins which bind to the intracellular domains of the p55 and p75 TNF-Rs and the Fas-R, which are capable of modulating the function of the p55 and p75 TNF-Rs and the Fas-R, and the DNA sequences which encode them. The present invention also concerns new soluble oligomeric TNF-Rs, oligomeric Fas-Rs and oligomeric receptors having a mixture of TNF-Rs and Fas-Rs. In addition, the present invention concerns methods of preparation and uses of all of the aforementioned.

Description

TNF/NGF superfamily receptors and modulators of soluble oligomeric TNF/NGF superfamily receptors

This application is a divisional application of the application dated May 11, 1995, and the application number is CN No.95194095.3.

field of invention

The present invention generally relates to certain receptors in the TNF/NGF receptor superfamily, and the regulation of their biological functions. The TNF/NGF receptor superfamily includes such receptors as P55 and P75 tumor necrosis factor receptors (TNF-Rs) and FAS ligand receptors (also known as FAS/APOI or FAS-R, hereinafter referred to as FAS-R) wait. More specifically, the present invention relates to novel proteins that bind to the intracellular regions (IC) of P55 and P75TNF-Rs and Fas-R (these intracellular regions are called P55IC, P75IC and Fas-IC, respectively). The protein can regulate the function of P55 and P75TNF-Rs and Fas-R. One of the proteins capable of binding the P55IC of the overall P55-TNF-R is P55IC itself or a part of the molecule, such as the "death domain" (abbreviated as DD) of P55IC. Thus, the present invention also relates to a novel TNF-related effect that can be induced intracellularly by the intracellular domain (P55IC) of P55TNF-R or a fragment thereof in a ligand-independent (TNF)-independent manner. The present invention also relates to the preparation and use of these novel proteins, including P55 and P75 TNF-R binding proteins, and FAS-R binding proteins, referred to herein as P55IC binding proteins, P75IC binding proteins and FAS-IC binding proteins.

On the other hand, the present invention also relates to novel soluble oligomeric TNF-Rs, oligomeric FAS-Rs and oligomeric receptors containing mixtures of TNF-Rs and FAS-Rs, their uses and production methods.

Background of the Invention and Prior Art

Tumor necrosis factor (TNF-α) and lymphotoxin (TNF-β) (TNF mentioned below refers to TNF-α and TNF-β) are mainly multifunctional inflammatory cytokines formed by mononuclear macrophages (pro-inflammatory cytokine), which has multiple effects on cells (Wallach, D. (1986)): Interferon 7 (Ion Gresser ed.), PP83-122, Academic press, London; and Beutler and Cerami (1987)). TNF-α and TNF-β exert their effects by binding to specific cell surface receptors. Some of their actions may be beneficial to the organism: for example, they can destroy tumor cells and virus-infected cells, and increase the antibacterial activity of granulocytes. Thus, TNF can act on the defense system of the organism against tumors and infectious agents, as well as on the recovery of damage. Therefore, TNF can be used as an anti-tumor agent in which TNF binds to receptors on the surface of tumor cells and thereby triggers the receptors on the surface of tumor cells and thereby causes the death of the tumor cells. TNF can also be used as an anti-infective agent.

However, TNF-α and TNF-β also have deleterious effects. Excessive synthesis of TNF-[alpha] has been shown to play a major pathogenic role in several diseases. The action of TNF-[alpha] on the vasculature is now known to be a major cause of septic shock (Tracey et al., 1996). In some diseases, TNF can cause excessive weight loss (cachexia) by inhibiting the activity of adipocytes and causing anorexia, so TNF-α is called cachexin. TNF has also been described as a regulator of tissue necrosis in rheumatism (Beutler and Cerami, 1987) and as a master regulator of adverse reactions occurring in graft versus host reactions (Piquet et al., 1987). Furthermore, TNF is known to be involved in inflammation and many other diseases.

Two different, independently expressed receptors, P55TNF-Rs and P75TNF-Rs, can specifically bind to TNF-α and TNF-β, thereby triggering and/or mediating the above-mentioned biological effects of TNF. These two receptors have structurally distinct intracellular domains and therefore signal differently (see Hohmann et al., 1989; Engelmann et al., 1990; Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990; Nophar et al. ; 1990; Smith et al., 1990; and Heller et al., 1990). However, its cellular mechanism, such as various related proteins and possible other factors involved in the intracellular signaling of P55 and P75TNF-Rs, has yet to be elucidated (the following is the first description, this new type of protein that can bind to P75IC and P55IC The protein is the intracellular signal, which usually occurs after the ligand, namely TNF (α or β), binds to the receptor, triggering a cascade amplification reaction, and finally leading to the cell's response to TNF.

Regarding the above-mentioned cytocidal effect of TNF, in most of the cells studied so far, this effect should be mainly triggered by P55TNF-R. Antibodies against the extracellular region (ligand-binding region) of P55TNF-R are themselves capable of triggering a cytocidal effect (see EP412486), and thus the cytocidal effect is linked to the effectiveness of antibody-receptor binding and is considered to be The first step in the process of generating intracellular signaling. In addition, mutation studies (Brakebusch et al., 1992; Tartaglia et al., 1993) showed that the biological function of P55TNF-R is determined by the integrity of its intracellular region, so it was proposed that the cytocidal effect of TNF is due to the initiation of intracellular signaling It is the result of association between two or more intracellular domains of P55TNF-R. Moreover, TNF (α or β) appears as a homotrimer and, as proposed, binds to and cross-links receptor molecules via P55 TNF-R, i.e., causes aggregation of the receptor Aggregation induces intracellular signaling. The following describes how P55IC and P55DD self-associate and induce TNF-related effects in cells in a ligand-independent manner.

Another member of the TNF/NGF receptor superfamily is the FAS receptor (FAS-R), which is also called the Fas antigen, a cell surface protein that can be expressed in various tissues and binds to many cell surface receptors. bodies, including TNF-R and NGF-R, have homology. FAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991), and acts as a negative selector for autoreactive T cells, that is, FAS-R during T cell maturation R mediates apoptosis of T cells that recognize self-antigens. Mutations in the FAS-R gene (IPr) were also found to cause a lymphoproliferative disorder in mice that resembles the human autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunage et al., 1992). The ligand for FAS-R appears to be a cell-surface-bound molecule carried by killer T cells (or cytotoxic T lymphocytes—CTLs), so that when such CTLs contact cells bearing FAS-R, they Ability to induce apoptosis of cells carrying FAS-R. Further, a monoclonal antibody specific for FAS-R has been prepared, which can induce apoptosis in cells carrying FAS-R, including small cells transformed with cDNA encoding human FAS-R. Apoptosis of murine cells (Itoh et al., 1991).

It has also been found that, except for T lymphocytes, various other normal cells can express FAS-R on their surface, and can be killed through the triggering of this receptor. Uncontrolled induction of this killing process may play a role in tissue damage in certain diseases, such as hepatocellular destruction in acute hepatitis. Therefore, finding ways to inhibit the cytotoxic activity of FAS-R has great medical potential.

Conversely, since certain malignant cells and HIV-infected cells have also been found to carry FAS-R on their surface, antibodies against FAS-R or FAS-R ligands in these cells can be used to trigger FAS-R-mediated induced cytotoxicity and thereby provide a means to counteract such malignant or HIV-infected cells (see Itoh et al., 1991). Finding other ways to enhance the cytotoxic activity of FAS-R also has medical potential.

It has long been felt that there is a need to provide a way to regulate cellular responses to TNF (α or β) and FAS-R ligands, for example, in the pathological conditions mentioned above, TNF or FAS-R ligands are overexpressed, thus It is necessary to inhibit the cytocidal effect induced by TNF or FAS-R ligand; in other cases, such as in the process of wound healing, it is necessary to enhance the effect of TNF, or in tumor cells or HIV-infected cells, it is necessary to enhance the mediation of FAS-R. leading role.

The present inventors have proposed several methods (see for example European application numbers: EP186833, EP308378, EP398327 and EP412486) to modulate the deleterious effects of TNF. For example, by inhibiting the binding of TNF to its receptors with anti-TNF antibodies, or by using soluble TNF receptors (essentially the soluble extracellular domain of the receptors) to competitively inhibit the binding of TNF to cell surface bound TNF-Rs . Further, based on the fact that TNF must bind to its receptors to induce cellular effects, the inventors have proposed some ways to regulate TNF effects, namely by regulating the activity of TNF-Rs. Briefly, EP0568925 is about a method of modulating signal transduction and/or cleavage of TNF-Rs by which peptides or other molecules interact with the receptor itself or with effectors (the Proteins that interact with receptors) interact to regulate the normal function of TNF-Rs. The construction and identification of various P55TNF-Rs mutants (mutants) are described in EP0 568925, and these mutations occur in the extracellular region, transmembranal region, and intracellular region of P55TNF-R. In this way, some regions within the above-mentioned P55 TNF-R region have been shown to be essential for the function of the receptor, that is, for ligand (TNF) binding and subsequent signal transduction and ultimately TNF effects. Both are necessary for intracellular signaling responses. Further, many approaches have been described for isolating and identifying proteins, peptides or other genetic factors that can bind to various segments within the above TNF-R functional domains, and these proteins, peptides and other factors are related to the regulation of TNF-R activity. Many other methods are also proposed in EP0568925, including how to isolate and clone genes encoding these proteins and peptides; how to construct expression vectors for producing these proteins; how to prepare antibodies to these proteins or peptides that can interact with TNF-R Fragments, there are also methods to prepare peptide fragments that bind to different regions of TNF-R. However, in EP0568925, neither the actual proteins and peptides (such as P55TNF-R) that can bind to the intracellular region of TNF-Rs are described, nor how to use yeast two-hybrid method (yeast two-hybrid) to isolate and identify these proteins or peptides that bind to the intracellular domain of TNF-Rs. Also, so far, no protein or peptide capable of binding to the intracellular region of FAS-R has been reported.

Therefore, when the effect of TNF or FAS-R ligand needs to be inhibited, the quantity or activity of TNF-Rs or FAS-R on the cell surface needs to be reduced; and when the effect of TNF or FAS-R ligand needs to be enhanced, It is necessary to increase the quantity or activity of TNF-Rs or FAS-R. In order to achieve this purpose, the inventors have recently carried out sequencing analysis to the promoters (promoters) of P55TNF-R and P75TNF-R, and found a variety of main key sequence elements (sequence motifs), which have specific effects on various transcriptional regulators. Specifically, the expression of these TNF-Rs was also found to be regulated at the promoter level, that is, inhibition of promoter transcription to reduce the number of receptors and enhancement of promoter transcription to increase the number of receptors (see IL104355 and IL109633 and their corresponding Corresponding sections on unpublished EP and PCT). Corresponding studies on the regulation at the level of the FAS-R gene promoter are yet to be reported.

Further, it should be mentioned that, on the one hand, the tumor necrosis factor (TNF) receptor, and the structurally related FAS-R receptor, are known to trigger intracellular lytic activity when stimulated by ligands produced by leukocytes. causes its own death, however little is known about the trigger mechanism. Mutational studies have indicated that in FAS-R and the P55 TNF receptor (P55-R), expression of cytotoxic signals involves distinct segments of their intracellular domains (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993) . These segments, the so-called "death domains" have sequence homology. The "death domains" of FAS-R and P55-R tend to self-associate. Their self-association (self-association) apparently promotes the aggregation of receptors (aggregation) that is required to initiate signaling (discussed below, and see Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995), receptor overexpression will trigger ligand-independent signaling (as described below, and Boldin et al., 1995).

Thus, prior to the present invention, no one has provided proteins capable of modulating the action of ligands belonging to the TNF/NGF superfamily, such as TNF or FAS-R ligands, which act on cells by mediating intracellular signaling This signaling is likely to be dominated by the intracellular regions (ICs) of some receptors belonging to the TNF/NGF receptor superfamily, such as the intracellular regions of TNF-Rs, namely P55 and p75TNF-R intracellular domain (P55IC and P75IC, respectively) and FAS-IC.

Thus, one of the objects of the present invention is to provide proteins which are capable of binding to the intracellular regions of TNF-Rs and FAS-R which are now thought to be involved in the process of intracellular signaling which occurs due to the binding of TNF to It is caused by its receptor, or by the binding of FAS ligand to its receptor.

Another object of the present invention is to provide antagonists (antagonists) (such as antibodies) against these intracellular domain binding proteins (IC-binding proteins), which antagonists can be used to inhibit the signaling process, that is, when the IC- Binding proteins are used when they are positive effectors (ie, induce signaling); or are used to enhance the signaling process, ie, when these proteins are negative effectors (ie, inhibit signaling).

Yet another object of the present invention is to use these IC-binding proteins to isolate and identify other related proteins or effectors. These proteins and effectors are involved in downstream and upstream events of signal transduction and IC-binding proteins such as other TNF-Rs or related receptors must also be involved in these events.

Another object of the present invention is to use the above-mentioned IC-binding proteins as antigens to prepare corresponding polyclonal and/or monoclonal antibodies. These antibodies can also in turn be used to purify new IC-binding proteins of different origin, eg from cell extracts or transformed cell lines.

Furthermore, these antibodies can be used for diagnostic purposes, eg for the identification of diseases associated with a malfunction of cellular effects mediated by receptors belonging to the TNF/NGF receptor superfamily.

It is a further object of the present invention to provide pharmaceutical compositions comprising the above-mentioned IC-binding proteins, and pharmaceutical compositions comprising antagonists of the IC-binding proteins, for the treatment or prevention of TNF-induced or FAS ligand-induced disorders, These drugs can be used to enhance the TNF effect or the FAS ligand effect, or to inhibit the TNF effect or the FAS ligand effect, eg depending on the nature of the IC-binding protein or its antagonist contained in the drug.

Moreover, according to another object of the present invention, the present invention discloses other methods to eliminate or antagonize Endogenous or externally administered TNF or FAS-R ligands. In this connection it should be mentioned that there have been attempts to isolate and recombinantly produce a TNF-binding protein called TBP-1 which has been shown to antagonize the action of TNF. This antagonism is measured by measuring the reduction of the cellular activity of TNF and by measuring the interference with the binding of TNF to its receptor (EP308378). TBP-1 has been shown to protect cells from the toxic effects of TNF and to interfere with the binding of TNF-α and TNF-β to cells at concentrations of several ng/ml in combination with these cytokines. Further research on the mechanism of action of TBP-1 shows that TBP-1 does not interact with target cells, but blocks the function of TNF by specifically binding to TNF, thereby competing with TNF receptors for TNF. Therefore, using different purification techniques, it is found that There are two active components: one is TBP-1 and the second is TNF-binding protein, which we call TBP-II (first described in EP398327). These two proteins can play a protective role against the cytocidal effect of TNF in vitro, and they bind TNF-β less effectively than TNF-α. Although the two proteins (TBP-I and TBP-II) appear to have very similar molecular weights by SDS gel electrophoresis, they can be clearly distinguished based on their lack of immunological cross-reactivity with each other, In addition, their N-terminal amino acid sequence and amino acid composition are different.

However, the above-mentioned early soluble TNF-binding proteins were monomers, and could only bind one monomer in the homotrimer, that is, one natural ligand, so that since TNF- still had two active monomers not bound by TNF Protein bound, TNF remains active (ie not fully neutralized). Moreover, there has been no report so far on soluble FAS-Rs (soluble FAS-R ligand-binding proteins), which are capable of binding to FAS-R ligands, and FAS-R is known to be a homologous Trimer, also a cell surface association molecule.

A so-called "death domain" of p55-IC (Tartaglia et al., 1993) has been discovered but not described. As described in the present invention, p55-IC and its "death domain" can be used to produce self-association, which is the main cause of signal generation and leads to the induction of cytotoxicity. Moreover, this report does not mention the possibility of synthesizing soluble oligomeric TFN-Rs, or soluble oligomeric FAS-Rs, or their mixed oligomers, nor is it revealed that other TNF-related effects, namely, mentioned by the present invention P55-IC or the effects it induces, eg IL-8-gene expression-induced effects. Similarly, another report, published after the date of the present invention, reported the aggregation (i.e., self-association) of P55-IC, but did not discuss the use of P55-IC self-association as described in the present invention. Cooperative production of soluble oligomeric TNF-Rs or FAS-Rs, nor other TNF-associated effects induced by p55-IC or parts thereof in a ligand-independent manner as described in the present invention.

Summary of the invention

As described in the present invention, we have discovered some novel proteins that either bind to the intracellular region of P55TNF-R (P55IC-binding protein), the intracellular region of P75TNF-R (P75IC-binding protein), or bind to In the intracellular domain of FAS-R (FAS-IC-binding protein). These P55IC-, P75IC- and FAS-IC-binding proteins can be used as mediators or modulators for the coordination effect of TNF or FAS-R on cells. Internal signaling process, which usually occurs after TNF binds to P55 and/or P75 TNF-R, or FAS-R ligand binds to the cell surface. And surprisingly, P55IC and FAS-IC can self-associate, and some fragments of P55IC and FAS-IC can also bind to P55IC, especially to the so-called "death domain" in these receptor ICS (DD), namely P55DD and FAS-DD. P55IC and PAS-IC, and fragments thereof, therefore also represent proteins that are able to bind to P55IC and FAS-IC and thus act as modulators of the ligand action of TNF or FAS-R on cells.

The nature of one of the novel proteins mentioned in the present invention, here named 55.11 protein, binding to the intracellular region of P55-TNF-R was further fully elucidated (see Example 1).

And, in another aspect, the invention is based on the discovery that the intracellular region of the P55 TNF receptor (P55-IC), is a region that contains the so-called "death domain" of the P55-IC; The intracellular domain of the body (Fas-IC), a region containing the so-called "death domain" of Fas-IC, is capable of self-association. Thereby it is possible to make a kind of soluble oligomeric TNF receptor according to conventional recombinant DNA technology, i.e. a kind of fusion protein, its one end contains at least two ectoregions of TNF receptor, and the other end contains at least two above-mentioned self-associated cellular domains. Inner regions, or a segment, are regions capable of self-association to provide an oligomer having at least two such fusion products linked together. Such soluble oligomeric TNF-R can bind two monomers in the naturally occurring TNF homotrimer, thereby effectively neutralizing the activity of TNF. Neutralization of TNF activity is necessary in all of the above-mentioned conditions, ie when TNF is produced in excess in vivo, or when administered in vitro at high doses, leading to undesired side effects. Furthermore, using the soluble oligomerization receptor of the present invention to effectively bind TNF can also be used to bind exogenous TNF, and then release slowly in a required amount, such as its application in the treatment of tumors. Likewise, according to conventional recombinant DNA techniques, it is also possible to construct an oligomeric FAS-R fusion product containing at least two extracellular domains of FAS-R at one end and at least two of the aforementioned self-associated intracellular domains at the other end. Regions or fragments thereof that self-associate to produce an oligomer in which at least two fusion products are linked together. This FAS-R oligomer can bind two monomers in the naturally occurring FAS-R ligand homotrimer, thereby effectively neutralizing the activity of the FAS-R ligand. Neutralization of the activity of the FAS-R ligand is necessary under all of the above conditions, since in these cases excess FAS-R ligand is associated with undesirable side effects. Likewise, given recent reports pointing to a possible link between TNF and FAS-R ligand-induced effects on cells, they may also be linked on the cell surface where the receptor is bound. It would thus be possible to construct a complex oligomer receptor specific for both TNF and FAS-R ligands using conventional recombinant DNA techniques. This complex oligomer is a mixture of the above-mentioned fusion products, one end contains at least one extracellular region of TNF-R and at least one extracellular region of FAS-R, and the other end contains at least two of the above-mentioned TNF-R and FAS- Self-associated intracellular regions of R, or fragments thereof, which are capable of self-association to produce a mixed oligomer in which at least two fusion products are linked together. Such a mixed oligomer can bind at least one monomer of TNF and at the same time bind a monomer of the FAS-R ligand, and this complex oligomer can also effectively reduce or neutralize TNF and FAS on the cell surface under the following conditions -R receptors, a condition in which excess of both hormones is associated with undesirable cellular effects as described above. As noted above, FAS-R ligands are usually cell surface associated and several recent reports describe cell surface-associated forms of TNF. These mixed TNF-R/FAS-R oligomers are therefore particularly effective for neutralizing the activity of TNF and FAS-R ligands on the cell surface.

To accommodate these circumstances, the present invention provides a DNA sequence encoding a protein capable of binding to one or more intracellular regions of one or more receptors belonging to the tumor necrosis factor/nerve growth factor ( TNF/NGF) receptor superfamily.

In particular, the present invention provides a DNA sequence selected from a large group of DNAs consisting of:

(a) a cDNA sequence derived from the coding segment of the native TNF-R intracellular domain binding protein;

(b) can hybridize to a certain section of DNA in (a) under less stringent conditions, and encode the DNA sequence of the TNF-R intracellular region-binding protein with biological activity; and

(c) Due to the degeneracy of the genetic code, the degenerate DNA sequences in (a) and (b) encode biologically active TNF-R intracellular region-binding proteins.

The present invention also provides a DNA sequence, which is selected from a large class of DNA family consisting of:

(a) a cDNA sequence that is derived from the coding segment of the native FAS-R intracellular domain-binding protein;

(b) DNA sequences capable of hybridizing to the cDNA of (a) under mild conditions and encoding a biologically active FAS-R intracellular region-binding protein; and

(c) Some DNA sequences degenerated as the DNA sequences defined in (a) and (b) as a result of the degeneracy value of the genetic code, which encode biologically active FAS-R intracellular domain-binding proteins.

In the present invention, some DNA sequences encoding P55TNF-R, P75TNF-R and FAS-R intracellular domain binding proteins are also included, for example, DNAs named as proteins 55.1, 55.3, 55.11, 75.3, 75.16, F2, F9 and DD11 sequence.

The present invention also provides a protein encoded by any one of the above-mentioned sequences of the present invention, or an analog, or a derivative thereof, said proteins, analogs and derivatives thereof are capable of binding to one or more than one TNF- One or more intracellular regions of Rs or FAS-R. Examples of this aspect of the invention include proteins 55.1, 55.3, 55.11, 75.3, 75.16, F2, F9 and DD11, and analogs and derivatives thereof.

The present invention also provides some vectors (vectors) encoded as the above-mentioned proteins of the present invention, which contain the above-mentioned DNA sequences of the present invention, and can be expressed in suitable eukaryotic host cells or prokaryotic host cells, and also provide a Transformed eukaryotic or prokaryotic host cells containing such vectors; and methods of producing proteins, analogs or derivatives of the present invention, that is, culturing these transformed host cells under suitable expression conditions, and expressing said proteins Post-translational processing, cell expression products are extracted from transformed cell culture media or cell extracts.

On the other hand, the present invention also provides some antibodies or active derivatives or fragments thereof against the protein of the present invention and its analogs and derivatives.

Another aspect of the present invention provides various uses of the aforementioned DNA sequences or proteins, including:

(i) A method for modulating the effect of TNF or FAS-R ligands on TNF-R or FAS-R loaded cells. comprising treating cells with one or more proteins, analogs or derivatives selected from the group consisting of P55IC, P55DD, FAS-IC or FAS-DD proteins and analogs or derivatives thereof, all of the above Proteins are capable of binding to intracellular regions and regulating the activity of said TNF-R or FAS-R, wherein said cell treatment comprises introducing one or more proteins, analogs or derivatives in a suitable form for intracellular introduction cells, or introducing the DNA sequence encoding the protein, analog or derivative into said cells in the form of a suitable expression vector;

(ii) a method for modulating the action of TNF or FAS-R ligands on cells carrying TNF-R or FAS-R, comprising, according to the invention, treating said cell;

(iii) a method for modulating the effect of TNF or FAS-R ligands on TNF-R or FAS-R loaded cells comprising treating said cells with oligonucleotide sequences, oligonucleotides according to the invention An antisense sequence (antisense sequence) of at least part of the sequence encoding the sequence, or an antisense sequence encoding P55IC, P55DD, FAS-IC, or FAS-DD sequence, said oligonucleotide sequence can block at least one The expression of a TNF-R or FAS-R intracellular domain-binding protein;

(iv) A method for modulating the effect of a TNF or FAS-R ligand on a TNF-R or FAS-R loaded cell comprising:

(a) Construct a recombinant animal virus vector, carrying a DNA sequence encoding a viral coat protein, which can bind to a specific cell surface receptor; and carrying another sequence selected from an oligonucleotide sequence wherein at least part of the oligonucleotide sequence is the antisense sequence of the protein coding sequence in the present invention, and an antisense oligonucleotide sequence selected from a sequence encoding P55IC, P55DD, FAS-IC or FAS-DD, Said oligonucleotide sequence is capable of blocking the expression of at least one TNF-R or FAS-R intracellular domain-binding protein when it enters the cell through viral mediation; and

(b) infecting said cells with said vector in (a)

(v) A method for modulating the effect of TNF or FAS-R ligands on TNF-R or FAS-R loaded cells comprising treating said cells with a suitable vector encoding a ribozyme which There is a sequence, which is specific to the mRNA sequence encoding the protein, analog or derivative described in the present invention; it is also specific to the mRNA sequence encoding P55IC, P55DD, FAS-IC or FAS-DD. Said ribozyme sequence can interact with said mRNA sequence and can cleave said mRNA sequence, resulting in inhibition of the protein mentioned in the present invention, expression of analogs or derivatives, or inhibition of P55IC, P55DD , expression of FAS-IC or FAS-DD;

(vi) a method for treating tumor cells or HIV-infected cells, or other diseased cells, comprising:

(a) Construction of a recombinant animal virus vector carrying a DNA sequence encoding a viral coat protein capable of binding to tumor cell surface receptors or HIV-infected cell surface receptors, or to the cell surface of other diseased cells Receptor; also carry the coding fragment of the protein, analog or derivative mentioned in the present invention and carry the sequence encoding P55IC, P55DD, FAS-IC, FAS-DD, or the analog or derivative thereof. Said protein of the present invention, its analogue or derivative, and P55IC, P55DD, FAS-IC, FAS-DD, analogue or derivative, when expressed in said tumor cell or HIV-infected cell or other diseased cells When in cells, both can kill these cells; and

(b) Infecting said tumor cells or HIV-infected cells or other infected cells with said vector in (a).

(vii) A method for isolating and identifying proteins, factors or receptors capable of binding to intracellular domain binding proteins according to the present invention. Isolation and identification methods include the use of affinity chromatography in which the proteins are attached to a matrix in affinity chromatography, and these attached proteins in turn interact with certain proteins, factors, or receptors in cell extracts , the bound protein is then eluted, separated and analyzed;

(viii) A method for isolating and identifying proteins. According to the invention, these proteins are capable of binding to intracellular domain binding proteins. The method involves the use of a yeast two-hyhrid approach, wherein the sequence encoding the intracellular domain-binding protein is carried by a hybrid vector, and another sequence from a cDNA or genomic library is carried by a second hybrid. Vectors (secondhyhrid vector), these vectors are then used to transform yeast host cells. isolating these positively transformed cells and subsequently extracting said second hybrid vector to obtain a sequence encoding a protein that binds to said intracellular domain binding protein; and

(ix) A method for isolating and identifying a protein capable of binding to the intracellular region of TNF-Rs or FAS-R comprising the use of mild Southern hybridization (nonstringentsouthern hybridization) followed by PCR amplification. According to the present invention, one of the sequences or a part thereof is used as a probe in a cDNA or genomic library to fish out sequences with at least partial homology. A large number of protein clones with homology to the sequence can be obtained by amplifying the homologous sequence by PCR.

The present invention also provides a drug for regulating the effect of TNF or FAS ligand on cells, comprising any of the following substances as an active component: (i) a protein according to the present invention, or protein P55IC, P55DD , FAS-IC or FAS-DD, or its biologically active fragments, analogs, derivatives or mixtures thereof, (ii) a recombinant animal virus vector encoding a viral coat protein capable of binding to TNF-R or Cell receptors carried by FAS-R or specific receptors of tumor cells; and a sequence encoding a protein, analog or derivative mentioned in the present invention or encoding P55IC, P55DD, FAS-IC or FAS-DD protein (iii) a recombinant animal virus vector encoding the virus coat protein as described in (ii) above, and the antisense oligonucleotide sequence of the P55IC, P55DD, FAS-IC or FAS-DD coding sequence; and (iv ) A ribozyme-encoding vector, the ribozyme can interact with the mRNA sequence encoding the protein, analogue or derivative, etc. of the present invention and the mRNA sequence encoding P55IC, P55DD, FAS-IC or FAS-DD.

A specific example of the above aspects of the invention is the use of P55-IC or the DNA sequence encoding itself. This example is based on the fact that P55IC can induce TNF-related effects in cells in a ligand-independent (TNF)-independent manner. There is thus provided a method for inducing TNF-related effects in cells or tissues comprising treating said cells with one or more proteins or analogs or derivatives thereof, said proteins being selected from Such a class of proteins, which substantially all belong to the self-associated intracellular region (P55-IC) of P55TNF-R or a part thereof, and can induce said intracellular TNF effect in a ligand-independent manner, wherein all Said cell treatment includes introducing said more than one protein, analog or derivative into said cell in a form suitable for intracellular introduction, or introducing a segment encoding more than one protein, analog or derivative The DNA sequence is introduced into said cells with a suitable vector, wherein the vector can complete the process of embedding the target sequence into the target cell, and then the target sequence can be expressed in the target cell.

Embodiments of the above method of the present invention include:

(i) a method, wherein said cell treatment is to transfect cells with a recombinant animal virus vector, comprising the steps of:

(a) Construction of a recombinant animal virus vector carrying a sequence encoding a viral coat protein (ligand) capable of binding to specific cell surface receptors on the surface of the cells to be treated. The vector also carries a second sequence which encodes p55-IC, or fragments, analogs and derivatives thereof. These proteins are capable of self-association upon cellular expression and can induce a range of TNF-related effects; and

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

(ii) a method, wherein said induction of TNF effect in cells refers to the induction of IL-8 gene expression, said carrier carries a sequence which encodes substantially all of said P55-IC, or Parts, and their analogs and derivatives. When expressed in cells, they can self-associate and signal to induce IL-8 gene expression.

(iii) A method for treating tumor cells or virus-infected cells, or for increasing the antibacterial effect of granulocytes, wherein said viral vector carries a sequence encoding a viral ligand capable of binding to tumor cells, virus Specific cell surface receptor on infected cells and granulocytes. It also carries sequences encoding p55-IC or parts thereof, analogs and derivatives which, when expressed in tumor cells, virus-infected cells or granulocytes, induce TNF-associated effects leading to the death of these cells.

(iv) A method for treating tumors or derivatives which, when expressed in tumor cells, induce expression of IL-8 leading to tumor cell death. Cytocidal is through its "chemotactic activity", that is, attracting granulocytes and other lymphocytes to tumor cells to cause tumor cell death.

In this aspect of the invention there is also provided the use of the intracellular region of P55-R, parts, analogs and derivatives thereof (P55-IC) in treating cells, i.e. inducing TNF-association effects; and providing The following examples are given:

(i) P55-IC, parts, analogs and derivatives thereof, are used to treat cells to induce IL-8 gene expression.

(ii) P55-IC, its parts, analogues and derivatives are used to treat tumor cells and kill tumor cells by inducing IL-8 gene expression.

Further, in this aspect of the invention, there is provided a pharmaceutical composition capable of inducing TNF-related effects. Including P55-IC, its parts, analogs and derivatives as active ingredients, and a pharmaceutically acceptable carrier (carrier); and their following examples:

(i) A pharmaceutical composition capable of inducing TNF-related effects, comprising as an active ingredient a recombinant animal virus vector encoded as P55-IC, parts thereof, and analogs and derivatives thereof, and capable of binding to treated Cell surface protein protein.

(ii) A pharmaceutical composition which, when treating tumor cells, induces the expression of IL-8 and consequently kills the tumor cells.

In another aspect, the invention provides a soluble, oligomeric tumor necrosis factor receptor (TNF-R) comprising at least two self-associated fusion proteins, each fusion protein having: (a) ,- a TNF-binding region, which is derived from the extracellular region of TNF-R, analogs or derivatives, and they do not affect its self-association and can bind TNF; and (b) at its other end, a self- Association region from: (i) substantially all intracellular region of P55TNF-R (P55-IC), i.e. from approximately amino acid residue 206 up to 426 of the native P55TNF-R molecule (P55-R) amino acid residues; (ii) the death domain of P55-IC, from approximately amino acid residue 328 to approximately amino acid residue 426 of native P55-R; (iii) essentially the FAS/AP01 receptor (FAS-IC); (iv) the death domain of Fas-IC; and (v) (i)-(iv) can self-associate any analog, a part thereof or derived Objects in which two self-association proteins are capable of self-association only at their ends (b). Its (a) end can bind at least two TNF monomers, and each (a) end can bind one TNF monomer, and the salts and functional derivatives of the soluble oligomeric TNF-R.

Some embodiments of this aspect of the invention also include all combinations of the above (a) terminus and (b) terminus, as defined above, for example, a soluble oligomeric TNF-R comprising p55 as the extracellular domain -R extracellular domain, and P55-IC as a self-associated intracellular domain.

Further, a process for producing the soluble oligomer TNF-R of the present invention is also provided, comprising:

(a) Construct an expression vector, which encodes any component of the fusion protein, and each segment of its DNA sequence is derived from the clonal line of the extracellular region, analog or derivative DNA sequence of TNF-R; The clone lines of DNA sequences of IC, P55-IC death function domain, Fas-IC, Fas-IC death function domain, and their analogs or derivatives can be connected to each other to form a fusion protein sequence. And said fusion protein sequence can be embedded in said carrier under the regulation of transcription and translation regulatory sequence;

(b) introducing the vector in (a) into a suitable host cell in which said fusion protein is expressed, and

(c) purifying the fusion protein expressed in the host cell, the fusion protein being self-associated before, during or after purification and producing soluble oligomeric TNF-R.

The following content is further provided: a vector encoding the above-mentioned fusion protein, which is widely used in the above-mentioned method of the present invention; a host cell containing the above-mentioned vector; and a pharmaceutical composition, which contains soluble oligomeric TNF- R and its salts, functional derivatives and a mixture of the aforementioned substances, as active ingredients, they are combined with pathologically acceptable carriers; at the same time, some soluble oligomeric TNF-R and its salts, functional Derivatives, as well as mixtures of the aforementioned substances, are used to antagonize the harmful effects of TNF in mammals, especially for the treatment of such conditions, that is, excessive TNF due to endogenous or exogenous administration; or in another case, when When TNF is administered exogenously, it is used to maintain the beneficial effects of TNF in mammals for a long time.

According to the above-listed clauses of the present invention, it has also been found that it is possible to prepare a soluble oligomer Fas/AP01 receptor (Fas-R) for use in antagonizing the deleterious effects of the FAS ligand. Accordingly, the present invention provides in another aspect a soluble oligomeric Fas/APO1 receptor (Fas-R) comprising at least two self-associated fusion proteins, each fusion protein having: (a) At one end of it, is a Fas ligand binding region, which is an extracellular region from Fas-R, an analog or derivative thereof, which cannot self-associate but is capable of binding Fas ligand; and (b) At its other end, is a self-association region derived from: (i) essentially the entire intracellular region of P55TNF-R (P55-IC), i.e. from about The segment between amino acid residue 206 to about amino acid residue 426; (ii) the death domain of P55-IC, i.e. from about amino acid residue 328 to about amino acid 426 of native P55-R (iii) substantially the entire intracellular region (Fas-IC) of the Fas/AP01 receptor; (iv) the death domain of Fas-IC; and (v)(i)-( Analogues or derivatives of the proteins described in iv) which are capable of self-association, wherein at least two of the self-associated proteins are capable of self-association only at (b) ends, their ends are capable of binding At least two Fas ligand monomers, and each (a) end can bind one Fas ligand monomer; and soluble salts and functional derivatives of oligomeric Fas-R.

According to this aspect of the present invention, there is also provided a method for producing soluble oligomeric Fas-R, comprising:

(a) Construction of an expression vector that encodes any component of the fusion protein, the DNA sequences at each end of the fusion protein being derived from a DNA clone that encodes substantially all of Fas-R, its analogs or derivatives , and from DNA clones that encode substantially all of P55-IC, P55-IC death domain, Fas-IC, Fas-IC death domain, and analogs or derivatives of all of the foregoing , each end of which can be connected to each other to form a fusion protein coding sequence, and the fusion protein coding sequence is embedded in the vector under the control of transcription and translation regulatory sequences;

(b) introducing the vector of (a) into a suitable host cell in which said fusion protein is expressed; and

(c) Purification of the fusion protein expressed in the host cell, said fusion protein being capable of self-association to give soluble oligomeric Fas-R before, during or after the purification process.

Furthermore, an expression vector is also provided, which carries the fusion protein coding sequence encoding soluble oligomeric Fas-R, and is widely used in the above-mentioned process; also provides a host cell carrying the vector and some drugs Composition, the pharmaceutical composition contains soluble oligomeric Fas-R and its salts or derivatives or a mixture of any of the foregoing as active ingredients, which are combined with pathologically acceptable carriers. At the same time, it also provides a soluble oligomeric Fas-R and its salts or functional derivatives or a mixture of any of the aforementioned substances, which are used to antagonize the harmful effects of Fas ligands on mammals, especially for treating endogenous Some conditions in which Fas ligand is overformed by raw or exogenous administration.

In a similar manner to that described above for oligomeric TNF-Rs and oligomeric Fas-Rs, it is also possible to prepare mixed oligomers that have binding specificities for both TNF and Fas-R ligands. Accordingly, the present invention also provides a mixed oligomeric TNF-R/FAS-R comprising at least two self-associated fusion proteins, one of which is derived from the above TNF-specific fusion protein Either, the other fusion protein is derived from the aforementioned FAS-R ligand-specific fusion protein, thereby providing a mixed oligomer having at least one TNF-R extracellular domain and at least one FAS-R extracellular domain, These extracellular domains are associated together by virtue of the self-association of the respective intracellular domains with which they are associated. These mixed oligomeric receptors are prepared, as described above, by first preparing oligomeric TNF-Rs and oligomeric FAS-Rs, then mixing them together, and then selecting ligands that specifically bind to FAS-R according to conventional procedures. and TNF oligomers. Another way to make mixed oligomeric receptors, as described above, is to co-transfect appropriate host cells with vectors encoding any of the TNF-specific fusion proteins (soluble TNF- Rs) and encoding any one FAS-R ligand-specific fusion protein (soluble FAS-Rs). Then purify, express the fusion protein, and it can self-associate before, during or after purification to obtain some oligomeric receptors, and then select these oligomeric receptors that can bind to TNF and FAS-R ligands according to conventional procedures receptor.

Likewise, a pharmaceutical composition is also provided, comprising mixed oligomeric receptors, their salts or functional derivatives, or a mixture of the aforementioned substances as active ingredients, combined with a pharmacologically acceptable carrier. Also provided are mixed oligomer receptors, their salts or functional derivatives, or mixtures of any of the foregoing, for use in antagonizing the deleterious effects of TNF and FAS-R ligands in mammals, particularly for the treatment of Conditions in which excess TNF and FAS-R ligands are formed as a result of endogenous or exogenous administration, or are otherwise used to maintain the beneficial effects of TNF and/or FAS-R ligands in mammals for a long time, for example when When they are administered in vitro together with TNF and/or FAS-R ligand (in soluble form).

The present invention also provides other aspects and embodiments described in detail below.

It should be noted that the following terms used throughout: "modulation of cells by TNF" and "modulation of cells by Fas ligand" should be understood to include in vitro and in vivo treatments.

Brief description of the drawings

Fig. 1 a-c has described the partial basic nucleotide sequence of some cDNA clones of coding P55IC and P75IC-binding protein with diagram, wherein Fig. 1 (a) is the clone 55.11 sequence of coding P55IC-binding protein 55.11 (SEQ ID NO: 9); Fig. 1 (b) is the partial basic sequence (SEQ ID NO:10 and 11) of the clone 75.3 of coding P75IC-binding protein 75.3; And Fig. 1 (c) is the clone 75.16 of coding P75IC-binding protein P75.16 Part of the basic sequence (SEQ ID NO: 12 and 13); all of these are as described in Example 1; and Figure 1 (d) describes the amino acid deduced sequence of protein 55.11 (SEQ ID NO: 14), and protein 55.11 is obtained from Figure 1 The deduced nucleotide sequence of (a) is also described in Example 1.

Figure 2 depicts the results of a Northern blot assay showing that 55.11-specific mRNAs are present in a variety of cell lines tested, as described in Example 1.

Figures 3A and 3B are the results of autoradiography, which show that the protein encoding 55.11cDNA binds to the GST fusion protein containing part of P55-IC in vitro, wherein Figure 3A describes the binding of the full-length 55.11 protein (55.11 full-length) to The situation of various GST fusion proteins; FIG. 3B depicts the binding of the portion 55.11 fused to the FLAG octapeptide to various GST fusion proteins, all described in Example 1.

FIG. 4 is a graph showing the deduced amino acid sequence of the human 55.11 protein compared to related protein sequences from lower organisms, as described in Example 1. FIG.

Figure 5 is the result of Western blot detection, which is stained with anti-MBP polyclonal antiserum, which is to illustrate the self-association of P55IC. When performing SDS-PAGE gel electrophoresis, the chimeric protein P55IC- Western blots were obtained for the interaction of MBP with P55IC-GST (lanes 1-4), or the regulatory effect between the chimeric protein P55IC-MBP and GST alone, (lanes 5-8). As for the interaction between chimeric proteins, before SDS-PAGE electrophoresis, electrophoresis was carried out on glutathione-agarose gel, as described in Example 2.

Figure 6 is the results of phase contrast microscopy, illustrating the IC cytotoxic effect of full-length P55 in HTtal cells transfected with an expression vector encoding P55IC (right panel), and illustrating the inhibition of this cytotoxic effect, that is, by treatment with tetracycline The situation when the expression of the vector was blocked by the cells (left panel), as described in Example 2.

Figure 7 depicts the ligand-independent triggering of the cytocidal effect of Hela cells (human cervical cancer passage cells) transfected by P55-R or its intracellular region, or part of the intracellular region including the "death domain" . in

(i) on the far left of Figure 7, various DNA sequences encoding full-length P55-R, its intracellular region and part of the intracellular region are described, and this sequence is inserted into the vector for transfection of Hela cells;

(ii) The left and middle bar graphs illustrate the expression of TNF receptors in Hela cells, that is, the expression of each type of receptor in Hela cells shown on the far left in Figure 7, and the left bar graphs represent receptors The amount (ng/cell sample), the middle bar represents the amount of the acceptor expressed as the radioiodinated TNF bound to the transfected cells, and

(iii) The bar graph on the right illustrates the viability of Hela cells expressing various receptors; in all the bar graphs, some open columns represent cells transfected in the presence of tetracycline, while closed columns represent cells transfected in the presence of tetracycline. Cells transfected in the absence of tetracycline; see Example 2 for all descriptions herein.

Figure 8 illustrates the ligand-independent induction of the IL-8 gene, which is expressed in Hela cells transfected by P55-R or its intracellular region (P55IC), wherein Figure 8A represents the results of Northern blotting . The RNA was extracted from Hela cells treated with TNF or untreated (two rows of left lanes marked with control and TNF respectively), and extracted from Hela cells transfected with vectors encoding P55-R and P55-IC or extracted from As a control protein luciferase (respectively labeled with P55-IC, P55-R and Luc's other swimming lanes), the Hela cells were transfected respectively in the presence of tetracycline (+) or in the absence of (-). Therefore, there are two lanes for each transfection); where Figure 8B shows methylene blue staining of 18srRNA in each HeLa cell sample shown in Figure A; all of the above are found in Example 2.

Figure 9 (A and B) schematically depicts the ligand-independent triggering of cytocidal effects in Hela cells. Hela cells were transfected with P55R or its parts, or with FAS-IC, wherein Figure 9 describes the results of Hela cells infected with P55R or its parts, and Figure 9B describes the results of cells infected with FAS-IC.

Parts of P55R or FAS-IC used for transfection are shown schematically on the left in Figures 9A and B, while some experimental results are shown on the right, all of which are described in Example 2.

Figure 10 schematically depicts part of the basic nucleotide sequence (SEQ ID NO: 19 and 20) of a cDNA clone named "F2", which encodes a protein that can bind to P55IC and FAS-IC, As described in Example 3.

Figure 11 schematically depicts part of the basic nucleotide sequence (SEQ ID NO: 21-23) of a cDNA clone named "F9", which encodes a protein that can bind to P55IC and FAS-IC, As described in Example 3.

Figure 12 schematically depicts part of the basic nucleotide sequence (SEQ ID NO: 24) of a cDNA clone named "DD11", which encodes a gene that can bind to P55IC, especially P55DD and FAS-IC Protein, as described in Example 3.

Detailed description of the invention

In one aspect, the present invention relates to some novel proteins that can bind to the intracellular region of receptors, wherein these receptors belong to the TNF/NGF superfamily, such as TNF-Rs and FAS-R, thus these proteins can be considered as receptors of this superfamily. mediators or regulators of bodies such as TNF-Rs and FAS-R, and these receptors have a certain effect. These proteins refer to the protein (P55IC) that can bind to the intracellular region of P55TNF-R, for example, some proteins named 55.1, 55.3 and 55.11 here (Example 1) and some proteins encoded by cDNA clone F2, F9 and DD11 ( Example 3); proteins (P75.IC) that can bind to the intracellular region of P75TNF-R, such as some proteins designated as 75.3 and 75.16 (Example 1); and some proteins that can bind to the intracellular region of FAS-R (FAS-IC), such as some proteins encoded by cDNA clones F2, F9 and DD11 (Example 3). proteins 55.1 and 55.3 have been found to be parts or fragments of the intracellular region of P55TNF-R (P55IC); the other proteins 55.11, 75.3 and 75.16 represent proteins (75.3, 75.16) that have not been reported before the present invention, or Although they have been reported (55.11, see khan et al., 1992), their functions and other properties, especially their ability to bind to TNF-R, have not been described in any way (see Example 1 below). These new proteins encoded by cDNA clones F2, F9 and DD11 also represent proteins that have not been reported before, that is, their sequences have not been stored in the "group database" (GENEBANK) or "protein database" ( PROTEIN BANK).

Thus, the present invention relates to the proteins encoded by these sequences, as well as the DNA sequences encoding these proteins.

Furthermore, the present invention also relates to DNA sequences encoding biologically active analogs and derivatives of these proteins, as well as analogs and derivatives encoded by them. These analogs and derivatives are prepared according to routine procedures (see for example Sambrook et al., 1989) in which one or more codons are deleted, added or replaced by another codon in the DNA sequence encoding these proteins , to obtain some analogs, which have at least one amino acid residue change compared with the native protein. A favored analog such as P55IC, P75IC or FAS-IC must retain at least the ability to bind to the intracellular domain of the TNF/NGF receptor superfamily, such as the intracellular domain of FAS-R or TNF-R, or be able to mediate Any other binding process or enzymatic activity, such as analogs that bind P55IC, P75IC or FAS-IC, but do not generate a signal, that is, cannot further bind downstream receptors (further downstream receptors), proteins or other Factors, or inability to catalyze a signal-dependent reaction. In this way it is possible to produce analogs which have a so-called dominant-negative effect, i.e. upon binding to e.g. process, the analogs are defective. These analogs can be used to inhibit the action of TNF or the action of FAS-ligand by competing with native IC-binding protein for binding. Likewise, so-called dominant-positive analogs can also be produced, which can be used to enhance the TNF effect or the FAS complexing effect. They have the same or better IC-binding properties, and the same or better signaling response properties than native IC-binding proteins. Similarly, some derivatives can also be prepared by changing the side groups of one or more amino acid residues of the protein by conventional modification techniques, or by conjugating the protein to another molecule, such as an antibody, enzyme, receptor, etc., as described in Described in the prior art.

These novel TNF-R and FAS-R intracellular domain binding proteins, such as proteins 55.1, 55.3, 55.11, 75.3, 75.16 and proteins encoded by cDNA clones F2, F9 and DD11 (hereinafter referred to as F2, F9 and DD11), all have Many possible uses such as:

(i) When it is necessary to increase the effect of TNF or FAS-R ligands, for example, when clinical applications such as anti-tumor, anti-inflammatory or anti-HIV require TNF or FAS-R ligands to induce cytotoxicity, they can be used to simulate or Enhance the function of TNF or FAS-R ligand. Proteins in this case, such as proteins that can bind to P55IC, like 55.1, 55.3, and proteins such as F2, F9 and DD11, as well as free P55IC itself that can enhance the effect of TNF (see below and Example 2), and P55IC's "Death domain", or proteins F2, F9 and DD11, FAS-IC and FAS-DD, which can enhance the coordination effect of FAS-R, ie the cytotoxic effect, can be introduced into cells according to known conventional methods. For example, since these proteins are intracellular and their introduction into cells requiring TNF effects or FAS-R coordination effects is desirable, a system for specifically introducing these proteins into cells is desired . One method for this purpose is to create a recombinant animal virus, such as a vaccinia virus, and introduce the following two genes into its DNA: one is a gene encoding a ligand that can bind to Specifically expressed cell surface proteins, such as AIDs (HIV) virus gp120 protein, can specifically bind to certain cells (CD4 lymphocytes and related leukemia), or can specifically bind to cells carrying TNF-R or FAS- Any ligand for cells of R, so that the recombinant viral vector can bind to cells carrying TNF-R or FAS-R; the other is encoding a novel intracellular domain-binding protein or P55IC, P55DD, FAS-IC or Gene for the FAS-DD protein. In this way, the expression of cell surface binding proteins on the surface of the virus will allow the virus to treat tumor cytotoxicity or other cells carrying TNF-R or FAS-R as specific target cells, and then the virus can bind to the intracellular region Protein coding sequences or P55IC, P55DD, FAS-IC or FAS-DD coding sequences are introduced into cells, and once they are expressed in cells, they can enhance the TNF effect or FAS-R coordination effect and lead to the death of tumor cells or cause the loading of tumor cells. Death of cells with TNF-R or FAS-R. Such recombinant animal viruses can be constructed following routine procedures (see, eg, Sambrook et al., 1989). Another possibility is to introduce novel protein coding sequences or P55IC, P55DD, FAS-IC or FAS-DD into cells in the form of oligonucleotide sequences and allow them to be expressed in cells.

(ii) They can be used to inhibit the action of TNF or FAS-R ligands. For example, in tissue damage due to septicemia, graft-vs-host rejection, or acute hepatitis, blocking TNF-induced TNF-R or FAS-R ligand-induced Intracellular signaling process of FAS-R. For example, introducing antisense (anti-sense) sequences encoding novel proteins, or antisense oligonucleotide sequences encoding P55IC, P55DD, FAS-IC or FAS-DD into cells by conventional methods can effectively block Translation and expression of mRNAs encoding proteins, ultimately inhibiting TNF effects or FAS-R coordination effects.

Such oligonucleotides can be introduced into cells by the above-mentioned recombinant virus method, that is, the virus carries the second oligonucleotide sequence. Another possibility is to use antibodies specific for these proteins to inhibit their intracellular signaling activity. There is also the possibility that these novel proteins have an extracellular region and an intracellular region, the intracellular region can bind to the binding region of TNF-R or FAS-R, and the antibodies produced can bind to their extracellular region, thereby Block their TNF-related functions or FAS-R ligand-related functions.

Yet another approach to inhibit the TNF effect or the FAS-R coordination effect follows the recently developed ribozyme approach. Nucleic acid is a catalytically active RNA molecule that can specifically cut RNA molecules, so it can selectively hydrolyze target RNAs, such as mRNAs encoding the novel protein of the present invention, or encoding P55IC, P55DD, FAS-IC or FAS -DD mRNA can be used as target RNAs. This ribozyme can recognize a specific sequence of a specific mRNA, and can interact with it (complementary binding) after mRNA cleavage, resulting in a reduction (or complete disappearance) of the expression of the protein to be inhibited, the degree of reduction (level), Depends on the degree of expression of the ribozyme in the target cell. In order to introduce ribozymes into target cells (such as cells carrying TNF-Rs or FAS-R), various suitable vectors can be used, such as plasmids, animal virus (retroviral) vectors. (See (i), wherein the virus has the cDNA sequence encoding the ribozyme of interest as the second sequence). Moreover, ribozymes are constructed to have multiple target substrates (multi-target ribozymes), which can be used to inhibit the expression of various proteins in the present invention and or the expression of proteins such as P55IC, P55DD, FAS-IC or FAS-DD (related For a review of ribozymes, methods, etc., see chen et al., 1992; Zhao and pick, 1993; Shore et al., 1993; Joseph and Burke, 1993; and Koizumi et al., 1993).

(iii) They can be used to isolate, identify and clone some other proteins that bind to the intracellular signaling processes downstream of TNF-R or FAS-R intracellular domains. In this case, the DNA sequences encoding these proteins can be used in the yeast two-hybrid system (see Example 1 below), i.e. these sequences will be used as "baits" from cDNA libraries or Genomic library to isolate, clone and identify the protein coding sequence that can bind to the intracellular region binding protein of these novel TNF-R or FAS-R.In this way, it is possible to determine the specific protein of the present invention, that is, bind to Whether the protein of P55IC, P75IC, or FAS-IC can bind to other receptors of the TNF/NGF receptor superfamily, for example, recently reported (schwalb et al., 1993; Baen et al., 1993; Crowe et al., 1994), in addition to P55 There are also other TNF-Rs in addition to P75TNF-Rs.Therefore, whether some proteins of the present invention can be specifically combined with other TNF-Rs or other receptors of the TNF/NGF superfamily can be specifically tested by using the yeast two-hybrid system Furthermore, this method can also be used to determine whether certain proteins of the invention are capable of binding to other known functionally active receptors.

(iv) These new proteins can be used to isolate, identify and clone other proteins of the same genus, that is, receptors that can bind to the intracellular region of TNF-R or FAS-R or functionally related receptors, as well as those related to the process of intracellular signal generation some protein. In this application, the yeast two-hybrid system described above can be used, or a more recently developed (wilks et al., 1989) system using non-stringent southern hybridization followed by PCR amplification can be used. In the article of Wilks et al., two recognized identification and cloning methods of tyrosine protein kinase (kinase) were described. Stringent Southern hybridization followed by PCR amplification. Utilizing novel protein sequences according to the present invention, this method can be used to identify and clone protein sequences related to TNF-R, FAS-R, or sequences related to receptor (TNF/NGF superfamily receptor) intracellular region binding proteins .

(v) There is another way to use the novel proteins of the present invention, that is, to use them in affinity chromatography to separate and identify other proteins or factors that can bind to them, such as separating and identifying proteins related to TNF-Rs (TNF /NGF receptor superfamily), or other proteins or factors involved in the process of intracellular signal generation. In this application, the novel proteins of the invention can be individually attached to an affinity chromatography matrix and then brought into contact with cell extracts or isolated proteins or factors presumed to be involved in intracellular signaling processes . With the progress of affinity chromatography, other proteins or factors bound to the novel protein of the present invention can be eluted, separated and identified.

(vi) As indicated above, the novel proteins of the present invention can also be used as immunogens (antigens) to produce antibodies specific to them. These antibodies can also be used to purify these novel proteins from cell extracts or transformed cell lines that produce them. Furthermore, these antibodies can be used for diagnostic purposes, ie for identifying diseases associated with malfunctioning of the TNF or FAS-R ligand system, eg cellular effects induced by hyperactive or hypoactive TNF or FAS-R ligands. Therefore, these antibodies could serve as an important tool for disease diagnosis if these diseases are associated with dysfunctional intracellular signaling systems of novel proteins.

It should also be noted that any known conventional screening method can be used for the isolation, identification and characterization of the novel proteins of the present invention. For example, the yeast two-hybrid method, described in the following examples (Example 1 and Example 3), was used to identify novel proteins of the invention. Also as described above and below, other methods, such as affinity chromatography, DNA hybridization, can also be used to isolate, identify and characterize novel proteins of the present invention, or to isolate, identify and characterize complementary proteins, factors, receptors These proteins, factors, and receptors can bind to the novel protein of the present invention or to some receptors in the TNF/NGF receptor family.

With respect to antibodies mentioned in the context herein, the term "antibody" includes polyclonal antibodies, monoclonal antibodies (mABs), chimeric antibodies, anti-idiotypic (abbreviated as anti-Id) antibodies to some antibodies, They can be labeled in soluble or bound form or in the form of fragments produced by known techniques. For example, it can be labeled by enzymatic cleavage, peptide synthesis or recombinant technology.

Polyclonal antibodies are heterogeneous antibody molecules derived from the serum of animals immunized with an antigen. Monoclonal antibodies include a class of homologous antibodies specific for certain antigens, ie they have substantially similar epitope binding sites. MAbs (monoclonal antibodies) can be prepared by methods well known to those skilled in the art. See, eg, Kohler and Milstein, Nature, 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al., eds., Harlow and Lan ANTIBODIES: A Laboratory Manual, Cold Spring Harbor Laboratory (1988); and Coligan, et al., eds., Immunology General Procedures, Greenes Publishing Assoc. and Wiley Interscience N.Y., (1992, 1993), the contents of these articles are fully incorporated into the specification by reference. These antibodies may belong to the IgG, IgM, IgE, IgA, GILD immunoglobulin families and subfamilies thereof. A hybridoma producing a mAb of the invention can be cultured in vitro, in situ or in vivo. In vivo or in situ cultures are capable of producing high titer mAbs, so they are currently the preferred methods for producing such mAbs.

Chimeric antibodies are antibody molecules in which different parts are derived from different animal species, for example, a class of antibody molecules whose variable regions are derived from murine mAbs and human immunoglobulin constant regions. Chimeric antibodies are mainly used to reduce immunogenicity and increase production yield. For example, mouse mAbs have a higher yield from hybridomas, but have higher immunogenicity in humans, so human/mouse chimeric mAbs are frequently used chimeric antibodies. The production method of the chimeric antibody is well known in the art (Cabilly et al., Proc.Natl Acad.Sci.USA 81:3273-3277 (1984); Morrison et al., Proc.Natl.Acad.Sci.USA 81 : 6851-6855 (1984); Boulianne et al., Nature 312: 643-646 (1984); Cabilly et al., European Patent Application 125023 (Published November 14, 1984); Neuberger et al., Nature 314: 268-270 (1985); Taniguchi et al. ，European Patent Application 171496(PublishedFebruary 19，1985)；Morrison等，European Patent Application173494(Published March 5，1986)；Neuberger等，PCT Application WO8601533(Published March 13，1986)；Kudo等，European PatentApplication 184187(Published June 11 , 1986); Sahagan et al, J.Immunol.137:1066-1074 (1986); Robinson et al, International Patent Application NO.WO8702671 (Published May 7, 1987); Liu et al, Proc.Natl.ACad.Sci.USA 84: 3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988); Manual, Supta. These articles are fully incorporated into the specification as a bibliography.

Anti-specific (Anti-idiotypic, abbreviated as Anti-Id) antibody refers to the ability to recognize idiotype determinants (determinants) associated with the antigen-binding site of the antibody. Id antibodies can be produced by immunizing an animal of the same species and genotype as the source of the MAb (for example, a mouse species) with MAb. The immunized animal recognizes or responds to the immunizing antibodies by producing an antibody (anti-Id antibody) specific for the idiotype determinant. See, eg, US Patent No. 4,699,880, which is incorporated herein by reference in its entirety.

Anti-Id antibody can also be used as an "immunogen" (immunogen) to induce an immune response in another animal, producing so-called anti-anti-Id (anti-anti-Id) antibody. This anti-anti-Id may be epitopically identical to the original mAb that elicited anti-Id. Thus, using antibodies raised against specific determinants of a mAb, it is possible to identify other clones expressing antibodies of the same specificity.

Thus, mAbs against IC-binding proteins of the invention, and analogs or derivatives thereof, or mAbs against P55IC, P55DD, FAS-IC, FAS-DD, and analogs or derivatives thereof can be used in suitable animals such as Induction of anti-Id antibodies in BALB/C mice. Spleen cells from these immunized mice can be used to produce anti-Id mAbs secreted by anti-Id hybridomas. Further, anti-Id mAbs can be linked to a carrier such as Keyholelimpet hemocyanin (KLH) and used to immunize supplemented BALB/C mice. The sera generated from these mice will contain anti-anti-Id antibodies that have the binding properties of the naive mAbs to the aforementioned IC-binding proteins, their analogs or derivatives, or P55IC, P55DD, FAS-IC or The antigenic epitopes of FAS-DD and its analogues or derivatives are specific.

Anti-Id mAbs thus have their own specific antigenic epitopes, or "idiotopes". They are structurally similar to the antigenic epitopes to be evaluated, such as GRB protein-alpha.

The term "antibody" includes both intact antibody molecules and antigen-binding fragments, such as Fab and F(ab') _{2 .} Clears faster than intact antibodies and has less non-specific tissue binding (Wahl et al., J. Nucl. Med. 24:316-325 (1983).

It is worth mentioning that Fab and F(ab') ₂ and other fragments of some of the antibodies used in the present invention can be used to detect and quantify IC-binding proteins or P55IC following the methods disclosed here for intact antibody molecules. , P55DD, FAS-IC, or FAS-DD. These antibody fragments are typically produced by hydrolysis of proteins with proteolytic enzymes such as papain (to produce Fab fragments), or pepsin (to produce F(ab') ₂ fragments).

If an antibody can specifically react with a molecule so that it binds to the antibody, we say that the antibody can bind a molecule. The term (antigen) epitope means a fragment of any molecule capable of being bound by an antibody and also recognized by an antibody. Epitopes or "antigenic determinants" usually refer to some surface chemically active groups such as amino acids or sugar side chains and have special three-dimensional structures and special charge characteristics.

An antigen is a molecule or a part of a molecule which is capable of being bound by an antibody and further capable of inducing an animal to produce antibodies which bind to an epitope of the corresponding antigen. An antigen can have one or more than one epitope. The above-mentioned specific reaction means that an antigen reacts with its corresponding antibody in a highly selective manner and does not react with antibodies elicited by other antigens.

The antibodies used in the present invention, including fragments of these antibodies, can be used to quantitatively or qualitatively detect IC-binding proteins or P55IC, P55DD, FAS-IC, FAS-DD in a sample, or to detect expression of the present invention. Cellular presence of IC-binding protein or P55IC, P55DD, FAS-IC, FAS-DD protein. Detection can be accomplished by immunofluorescence, ie, the use of fluorescently labeled antibodies (see below) in combination with detection techniques such as light microscopy, flow cytometry, or fluorescence detection.

Certain antibodies and fragments thereof useful in the present invention can be used to detect IC-binding proteins of the present invention or P55IC-P55DD, P55IC-P55DD, FAS-IC, FAS-DD. In situ detection is accomplished by cutting tissue samples from patients and adding the labeled antibody of the present invention. The best way to add antibodies (or fragments thereof) is to attach or superimpose labeled antibodies (or fragments) on tissue samples. By this, it is possible not only to determine the presence of the IC-binding protein or P55IC, P55DD, FAS-IC, FAS-DD, but also its distribution on the investigated tissue. Utilizing the present invention, one of ordinary skill will also readily appreciate that any of a variety of histological methods, such as staining, can be modified for in situ detection.

Assays for IC-binding proteins or P55IC, P55DD, FAS-IC, FAS-DD for use in the present invention typically comprise, first in an available assay capable of identifying the IC-binding protein or P55IC, P55IC, FAS-IC, FAS-DD In the presence of the labeled antibody, culture samples, such as biological fluids, tissue extracts, freshly collected cells such as lymphocytes or leukocytes, or cells that have been grown in tissue culture, and then use any known technique to detect the antibody .

Biological samples may be treated with a solid support or carrier such as nitrocellulose, or other solid support or carrier capable of immobilizing cells, cell particles, or soluble proteins. These supports or carriers are then washed with a suitable buffer and subsequently treated with a detectably labeled antibody, according to the invention, as described above. The solid support or carrier is then washed a second time with buffer to remove unbound antibody. The amount of the label bound to the solid support or carrier can be detected by ordinary methods.

The term "solid phase support", "solid phase carrier", "solid support", "solid carrier", "support" or "carrier" shall be extended to any support capable of binding antigen or antibody or carrier. Known supports or carriers, including glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, magnetic iron ore. The nature of the carrier can be soluble or insoluble to some extent. The support material can be of virtually any possible structural configuration as long as it enables the attached molecule to bind to the antigen or antibody. Thus, the configuration of the support or carrier may be spherical, such as a bead, or cylindrical, for example placed inside a test tube, or placed on the outside of a bar. Alternatively, their surface may be flat, such as in the form of a sheet, test strip, or the like. A preferred support or carrier is polystyrene beads. Those skilled in the art will know that many other suitable carriers can be used to bind the antibody or antigen, or can ascertain the same by using routine experimentation.

The binding activities of many of the above-mentioned antibodies of the present invention can be determined according to known methods. Those skilled in the art can determine the most suitable operating and assay conditions by routine experiments.

Other operating steps, such as washing, stirring, shaking, filtering, etc., can be increased or decreased according to the routine.

According to the present invention, one of the detection methods of the labeled antibody is to link the antibody to an enzyme and then use it in an enzyme immunoassay (EIA). The enzyme, when brought into contact with an appropriate substrate, reacts with the substrate to produce a chemical lysate which can be detected, for example, by spectrophotometric, fluorometric or objective methods. Measured by sight. Enzymes that can be used for labeled antibody detection include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5 steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase , triose phosphate isomerase, horseradish (horseradish) peroxidase, alkaline phosphatase, asparaginase, galactosidase, ribonuclease, urease, catalase, glucose starch Enzyme, acetylcholinesterase. Detection can be done colorimetrically, ie using a chromogenic enzyme substrate for the enzyme. Detection can also be accomplished by visual comparison, ie, the degree of enzymatic reaction of the enzyme on the substrate compared to a similarly prepared standard.

Detection can also be performed by any of the other immunoassays. For example, using radiolabeled various antibodies and antibody fragments, it is possible to detect RPTPase (R-PTPase) by using radioimmunoassay (abbreviated as RIA). For a better description of RIA, see work, T.S. et al.'s report in "Laboratory Techniques and Biochemistry in Molecular Biology", North Holland Publishing company, NY (1978), with specific reference to Chard, T's book titled Chapter "An Introduction to Radioimmune Assay and Related Techniques", this article has been included in the bibliography. Radioactive isotopes can also be detected using r counters or scintillation counters or by autoradiography.

According to the invention it is also possible to label antibodies with fluorescent compounds. When an antibody labeled with a fluorescent compound is exposed to light of the appropriate wavelength, its presence can be detected due to the generation of fluorescence. The most commonly used fluorescent labeling compounds are fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine.

Antibodies can also be made detectable labels using fluorescent radioactive metals such as 152E or other lanthanides. These metals can be linked to the antibody by using the chelating groups of these metals such as diethylenetriaminepentaacetic acid (ETPA).

Antibodies can also be made detectable labels by linking them to chemiluminescent compounds. Chemiluminescent-labeled antibodies are detected by detecting the bright light produced during the chemical reaction. Particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, and oxalates.

Likewise, bioluminescent compounds can also be used to label the antibodies of the invention. Bioluminescence is a type of chemiluminescent phenomenon that occurs in biological systems, in which a catalytic protein increases the efficiency of chemiluminescence. The presence of bioluminescent proteins can be detected by detecting the presence of bright light. Important bioluminescent compounds for labeling purposes are luciferin, luciferase, aequorin.

The antibody molecules of the present invention are suitable for use in immunoassays, also known as "two-site" or "sandwich" assays. In a typical immunoassay, an amount of unlabeled soluble antibody (or antibody fragment) is bound to a solid support or carrier, and a detectable amount of soluble labeled antibody is added, making it possible to detect and/or quantify the presence of A ternary complex formed between solid-phase antibody, antigen, and labeled antibody.

Typical and preferred immunoassays include "forward" assays, in which antibodies already bound to a solid phase are first contacted with the sample to be tested in order to pass through the formation of a binary solid phase antibody - Antigen complex, extracting antigen from the sample. After an appropriate incubation, the solid support or carrier is washed to remove residues of the fluid sample, including unreacted antigen (if any), and is bound to an unknown amount of labeled antibody (which acts as a "reporter"). molecule") solution contact. After the second incubation period, the labeled antibody is allowed to complex with the antigen bound to the solid support or carrier by the unlabeled antibody, and the solid support or carrier is then washed a second time to remove unreacted labeled antibody.

In another class of "sandwich" assays, so-called "simultaneous" and "reverse" assays are used, both "sandwich" assays are useful with the antigens of the invention. When the antibody bound to the solid support or solid carrier is added to the sample to be tested at the same time as the labeled antibody, the "simultaneous assay" involves a separate incubation step, after which the solid support or carrier is washed to remove fluid Residue of sample and uncomplexed labeled antibody. The labeled antibody associated with the solid support or carrier is then assayed following a common "forward" sandwich assay.

In "reverse" assays, a stepwise addition is used whereby labeled antibody is first added to a fluid sample, followed by unlabeled antibody bound to a solid support or carrier after an appropriate incubation period. After the second incubation, the solid phase is washed in the usual manner to remove residues of the sample to be tested and unreacted labeled antibody. The labeled antibody associated with the solid support or carrier is then assayed in "simultaneous" and "forward" assays.

The novel proteins of the present invention, once isolated, identified, and characterized by any conventional screening method, such as yeast two-hybrid method, affinity chromatography, and any known technique, can be produced in large quantities using conventional recombinant DNA techniques (for example, see Sambrook et al., 1989), that is, cells of appropriate eukaryotic or prokaryotic hosts are transformed with appropriate eukaryotic or prokaryotic vectors (containing sequences encoding proteins). Thus, the present invention also relates to such expression vectors and transformed host cells for the production of some of the proteins of the present invention. As mentioned above, these proteins also include their biologically active analogs and derivatives, as well as vectors with corresponding coding sequences and transformed host cells used to produce these analogs. The derivatives of these proteins refer to The modification method modifies the protein or its analog produced by the transformed host cell to produce the product.

In another aspect, the present invention also relates to the use of the intracellular region of P55TNF-R (P55IC) or the intracellular region of FAS-R (FAS-IC) or their so-called "death domains" (P55DD or FAS-DD respectively). ) as an enhancer (agentforenhancing) to enhance the effect of TNF or FAS-R ligand on cells, and the effect on itself (see Example 2). When TNF-induced or FAS-R ligand-induced cytotoxicity needs to be introduced somewhere, such as in cancer cells or HIV-infected cells, the above-mentioned (see (i) above) recombinant animal virus (such as vaccinia) approach is convenient. P55IC, P55DD, FAS-IC or FAS-DD can be introduced into these cells. Natural P55IC, P55DD, FAS-IC or FAS-DD, and biologically active analogues and derivatives or fragments thereof may also be used here, all of which may be prepared as described above.

Similarly, the present invention also relates to specific blocking (specific blocking) effect on TNF effect or FAS-R ligand effect, that is to block the activity of P55IC, P55DD, FAS-IC or FAS-DD, such as some antisense oligonuclear Nucleotide sequences can be introduced into these cells to block the expression of P55IC, P55DD, FAS-IC or FAS-DD.

The present invention also relates to some pharmaceutical compositions, which comprise some recombinant animal virus vectors encoding TNF-R or FAS-R intracellular region binding proteins (including P55IC, P55DD, FAS-IC and FAS-DD), these vectors also encode Specific target cell (such as cancer cell) surface protein to direct the intracellular domain binding protein sequence to embed virus surface protein in certain cells.

In another aspect, the present invention also details the effect of self-association of the intracellular domain of the P55 TNF receptor (P55IC, see Example 2). For example, the expression of the gene encoding IL-8, this effect is usually mediated by the binding of TNF to its receptor, and can also be mimicked by the signaling activity generated by the self-association of P55-IC or its fragments.

IL-8 is a cytokine (cytokine), which belongs to the chemokines (chemokines) subclass, has basic chemotactic activity (chemotactic activity) and has been proved in the granulosa cells and cells associated with many pathological conditions. plays an important role in the chemotaxis of granulocytes (see eg Endo et al., 1994; Sekido et al., 1993; Harada et al., 1993; Ferrick et al., 1991).

TNF has beneficial activities such as being used therapeutically to destroy tumor cells and virus-infected cells or to increase the antibacterial activity of granulosa cells. However, as noted above, TNF also has undesirable activities, and it is desirable to block these activities in situations including the use of high doses of TNF for cancer therapy, antiviral therapy, or antibacterial therapy, among others.

Therefore, it is hoped that it would be possible to direct TNF, or substances that mimic its beneficial activity, to the cells or tissues in need of treatment, which would be particularly beneficial therapeutically.

According to the present invention, it has now been found that the self-associated intracellular domain of P55-R (P55-IC) can mimic many effects of TNF in a ligand-independent manner, for example the "death domain" of P55-IC can Induce cytocidal effect, and P55-IC can induce IL-8 gene expression. Thus, it is possible to use p55-IC to mimic TNF function in a point-localized manner, ie to induce p55-IC to act only on cells or tissues that require treatment.

One of the above-mentioned approaches, as mentioned above, is to specifically transfect tumor cells or malignant tissues with DNA molecules encoding p55-IC or a part thereof, which can not only induce cytocidal effects on these cells or tissues, but also through the interaction with IL Co-induction of -8 enhances these effects, resulting in accumulation of granulosa cells and other lymphocytes on such cells or tissues, which in turn serve to kill tumor cells or tissues. This approach eliminates the potent killing side effects associated with high doses of TNF.

Using conventional recombinant DNA technology, it is possible to prepare various regions of p55-IC and determine which region is responsible for each TNF-induced effect. For example, we have determined that the "death domain" is related to the cell Toxicity is involved (Example 2), and various other constructs have been made that contain parts of the P55-IC that (along with some or all of the death domain) are responsible for other TNF-effects and that they can be independent of Ligands are used that, once self-associated, will induce these effects, such as the induction of IL-8 expression.

It should be noted that the sequence of the P55-IC related to the induction of other TNF-related effects is different from that of the P55-IC related to the induction of cytotoxicity, that is, it may not include or include part of the "death domain" and have Sequence motifs (motits) of other segments, or possibly also the same sequence, different features of this sequence (identical sequence motifs) are involved in the induction of different effects.

Thus, as detailed above and below, expression vectors containing p55-IC fragments, analogs or derivatives thereof can be prepared, expressed, purified and tested for their activity. In this way, a variety of P55-IC fragments with one or more TNF-related activities can be prepared and used in different ways to treat various diseases, such as viral infections, bacterial infections, tumors, and the like. In all these cases, the increase in specific activity can be achieved by binding (or co-transfection) with the p55-IC fragment, which is mainly responsible for the induction of IL-8 gene expression, and the chemotactic activity of IL-8 can Used to enhance the destructive effect on cells or tissues that need to be killed.

Thus, without systemic administration of TNF, it is possible to induce its beneficial effects by introducing all or part of the p55-IC into cells or tissues in need of treatment.

By any of the methods described above, p55-IC can be specifically introduced into cells or tissues desired to be killed. For example, one method is to construct a recombinant animal virus, such as a virus from vaccinia, and introduce the following two genes into its DNA: a ligand encoded by a gene can specifically bind to a cell surface protein, such as a specific AIDS virus gp120 protein that binds to certain cells (CD4 lymphocytes and related leukemia), or encodes a ligand that can specifically bind to cells carrying TNF-R, such recombinant virus vectors will be able to bind to some cells carrying TNF-R -R cells; another gene encoding p55-IC or a fragment thereof. In this way, the expression of cell surface binding proteins on the surface of the virus will allow the virus to target tumor cells or other cells carrying TNF-R, and then the coding sequence of p55-IC or a part thereof will be introduced into these cells by the virus. cell. And, once expressed in these cells, it will enhance the TNF effect and lead to the death of tumor cells or other TNF-R-loaded cells, or induce IL-8 and lead to the death of these cells. For the construction of such recombinant animal viruses, conventional techniques can be used (see, eg, Sambrook et al., 1989). Another possibility is to introduce the p55-IC sequence or parts thereof into the cells in the form of oligonucleotides which can be taken up by the cells and expressed therein.

Therefore, the present invention also specifically relates to some pharmaceutical compositions, comprising the above-mentioned recombinant animal virus vector encoding P55-IC or a part thereof. The coding sequences of p55-IC, or fragments thereof, are directed to insert into these cells.

Still another aspect of the present invention relates to novel synthetic TNF receptors that are soluble and capable of forming dimers and possibly higher-order multimeric TNF receptor molecules, each monomeric portion of which can Binds to a TNF monomer. Naturally occurring TNF is a homotrimer containing three active TNF monomers, each of which can bind to a TNF receptor molecule, while some naturally occurring TNF receptors are monomers, each of which is only Can bind a monomer in the TNF homotrimeric molecule. Thus, when TNF binds to some of the TNF receptors on the cell surface, it binds to three receptor molecules, resulting in a cluster of TNF receptors, which is thought to be the beginning of the signaling process that ultimately triggers the Observed TNF effects.

On the one hand, TNF has many beneficial effects, such as it can kill tumor cells or virus-infected cells and increase the antibacterial activity of granulosa cells, but TNF also has many adverse effects, such as in many serious diseases including autoimmune diseases, rheumatoid arthritis Inflammation, graft-versus-host reaction (transplant rejection), septic shock, etc., TNF is considered to be the main cause of pathological tissue damage. TNF also causes severe wasting (cachexia) due to the inhibition of adipocyte activity. Moreover, even when administered according to its desired activity, such as in the treatment of various malignant or viral diseases, the doses of TNF used are often high enough to cause many adverse cytotoxic side effects in patients, such as healthy tissue damage.

In all of the above cases, the action of TNF is unfavorable and therefore effective inhibitors of TNF have been sought. Many TNF-blocking agents (TNF-blocking agents) have been proposed, including some soluble proteins, which can bind TNF and inhibit TNF from binding to its receptors, thereby inhibiting the cytotoxic effect of TNF (see EP308378, EP398327 and EP568925 ). However, these TNF-binding proteins, or soluble TNF receptors, are monomers, each binding only one monomer in the TNF homotrimer. Therefore, the blockage of TNF function may not be complete, and the TNF molecule after each monomer receptor body binds still has two TNF monomers in a free state, and can also bind to TNF receptors on the cell surface, causing adverse side effects on cells .

In order to overcome the above-mentioned disadvantages when blocking TNF function, the present invention has developed a method for constructing some soluble oligomeric TNF receptors, that is, some fusion proteins, which are able to bind at least one of the naturally occurring TNF homotrimeric molecules. Two TNF monomers. As a result, these soluble oligomeric TNF receptors bind their TNF ligands more strongly than previously known monomeric soluble TNF binding proteins or receptors. For example, when the soluble TNF receptors of the present invention are in dimeric form, it is possible to bind both monomers of the TNF trimer, thereby causing more complete TNF neutralization. This neutralization is longer lasting due to the lower dissociation rate of soluble dimeric receptors from TNF. Furthermore, the larger molecular weight of such soluble oligomeric receptors compared to their monomeric counterparts is also of pharmacological advantage since they are likely to be cleared from the body at a slower rate.

The development of the soluble oligomeric TNF receptors of the present invention is based on the discovery that the intracellular domain of the P55-R TNF receptor is capable of self-association and the further discovery that within this intracellular domain (P55-IC) there is a segment, Called the "death domain", it is also self-associated and can produce cytotoxic effects in a ligand-independent manner (see Example 2). Utilizing the self-association property of P55-IC and its "death domain", through conventional recombinant DNA technology, it is possible to construct a fusion protein that contains the entire extracellular region of TNF receptors, such as P75-R Or some receptor of P55-R, preferably P55-R, fused to the whole intracellular region (P55-IC) or the death domain of P55-IC. This produces a novel fusion product that has the TNF binding domain, the extracellular domain of the receptor, at one end and the intracellular domain or its death domain at the other end, and is capable of self-association. Thus, polymerization occurs via self-association between two (and possibly more than two) p55-ICs or their death domains, resulting in some oligomers (or at least two aggregates).

Further, according to the present invention, it has also been found that the Fas/APO1 receptor also has self-association, and its intracellular region contains a self-association "death function domain", and has certain interactions with P55-IC and its death function domain. Homology (Example 2). It is thus possible to construct some of the soluble oligomeric TNF receptors of the invention by fusing the extracellular domain of the TNF receptor (as described above) with the intracellular domain or "death domain" of the Fas/AP01 receptor.

In both cases, the oligomeric TNF receptors of the invention, which are soluble, actually have only the soluble extracellular domain of the TNF receptor and the soluble cellular domain of both the P55-RTNF receptor or the Fas/APO1 receptor. The inner regions or their "death domains", ie they do not contain the transmembranal or insoluble regions of the two types of receptors.

The construction of the oligomeric TNF receptor of the present invention is described in detail in Example 4 below. Still need to point out, when constructing the oligomeric TNF acceptor of the present invention, also can produce a kind of situation, this kind of situation has not been reported before this, the extracellular region of TNF receptor can self-association, this kind of situation may be Unfavorable, because it will interfere with the binding of low oligomerization receptors to two or more TNF monomers of the homotrimeric molecule, or lead to weakened binding ability to TNF monomers. Thus, in this case, it is possible to modify the extracellular region of the TNF receptor using conventional recombinant DNA techniques, for example by deleting or substituting one or more amino acid residues in the self-association segment, to prevent such self-association. association phenomenon. Such modifications of the extracellular domain of TNF are also part of the present invention and are termed analogs or derivatives of the extracellular domain of TNF receptors. Likewise, the self-associated intracellular region (IC) of the P55-R receptor or the Fas/APO1 receptor or its death domain (DD), which have been used in the oligomeric TNF receptors of the present invention, may also be other Analogs or derivatives, that is, it can be a modified product of the P55-IC sequence or its fragment including the death domain (P55DD) or the Fas/APO1 intracellular region (FAS-IC) sequence or its death domain ( FASDD), if these modifications result in a self-association product.

Likewise, soluble oligomeric TNF receptors, their analogs or derivatives, once produced and purified, can be further modified using conventional chemical methods to provide their salts and functional derivatives for the preparation of some pharmaceutical combinations These pharmaceutical compositions all contain the TNF receptor of the present invention as an active ingredient.

To produce the soluble oligomeric TNF receptor of the present invention, the DNA sequence encoding the extracellular domain of the TNF receptor is obtained from all existing clones of the TNF receptor, as is the DNA encoding sequence for its intracellular domain or death domain , and the DNA coding sequence of the intracellular region or the death domain of the Fas/APO1 receptor (see Example 2 and Example 5). In this way, the DNA coding sequence for the desired extracellular region can be ligated to the DNA coding sequence for the desired intracellular region or a fragment thereof that includes the death domain. Under the control of , it is embedded (and connected) in an appropriate expression vector. Once formed, the expression vector is introduced (transformed, transfected, etc.) into an appropriate host cell, and the vector is then expressed to yield the fusion products of the invention, ie, certain soluble self-associated TNF receptor molecules. These molecules are then purified from the host cells using conventional procedures to yield the final product, soluble oligomeric TNF receptors.

A better preparation method for the fusion product encoding the extracellular region and the intracellular region or a part thereof is to use oligonucleotides specific to the desired sequence as probes according to the PCR technique route. The desired sequences were copied from clones encoding all TNF receptor molecules. Another approach is also possible, e.g. by restriction endonucleases isolating the desired parts encoding the extracellular and intracellular regions and then joining them together in a known manner, with modifications at the ends of the cleaved fragments Or not, to ensure proper fusion of the required parts of the receptor (extracellular and intracellular regions or parts thereof). The fusion product thus obtained is then inserted into the expression vector of choice.

In the same way, the present invention also relates to certain soluble oligomeric Fas/APO1 (FAS) receptors, which contain the extracellular region of the Fas/APO1 receptor and the self-associated intracellular region of P55-R (P55-IC) and The death domain thereof (P55DD), or the self-associated intracellular region of the Fas/AP01 receptor (FAS-IC) or its death domain (FASDD), or any analogue or derivative thereof (see above). The construction of these soluble oligomeric FAS receptors is detailed in Example 5 below, using the full-length FAS receptor-coding sequence as starting material and appropriate oligomeric primers for the desired extracellular and intracellular regions PCR amplification of the coding sequences, followed by ligation to obtain fusion products, which are inserted into appropriate expression vectors.

As detailed above and below, prokaryotic or eukaryotic cell vectors and host cells can be used to produce the desired soluble oligomeric FAS receptors, which are then purified and dispensed as active ingredients into pharmaceutical compositions.

The above-mentioned soluble oligomeric FAS receptors of the present invention are intended to effectively block Fas ligands, which also exist as trimers (similar to TNF, see above), and each oligomeric receptor of the present invention Each body is capable of binding two or possibly more Fas ligands and thereby neutralizes their activity. The Fas ligand is known to be primarily cell-surface associated, but also exists in soluble form. Regardless, the oligomeric FAS receptors of the invention are capable of binding to at least two monomers of such ligands and thereby neutralizing the activity of the Fas ligand more efficiently (than monomeric FAS receptors). Fas ligand, and the activation of FAS receptors, are involved in many diseases, especially those related to liver damage (such as apoptosis of liver cells, including hepatitis-related liver damage, and autoimmune symptoms, including Lymphocyte damage (programmed cell death) in HIV infection human body (for example see Ogasawara et al., 1993; Cheng et al., 1994).Therefore the soluble oligomeric FAS acceptor of the present invention is intended to block Fas ligand activity, and It can be used as an active ingredient of a pharmaceutical composition for treating diseases related to Fas ligand, for example.

Likewise, the present invention also relates to soluble oligomeric receptors that have affinity for both TNF and FAS-R ligands, these oligomeric receptors are so-called "mixed" TNF-R/FAS-R oligomeric receptors. body. These oligomeric receptors contain at least one TNF-R ectodomain and at least one FAS-R ectodomain, and they are capable of binding the aforementioned self-associated P55IC, One of P55DD, FASIC or FASDD is fused to combine with each other.

These mixed oligomer receptors can be prepared by (a) providing any of the above fusion products containing the extracellular domain of TNF-R (P75TNF-R, or preferably P55TNF-R) fused to any of the self- associating intracellular domains P55IC and FASIC or any one of the self-associated "death domains" P55DD and FASDD, or any self-associated fragment, any analogue or derivative thereof, (b) providing any of the above fusion products, They contain the extracellular domain of FAS-R fused to any one of self-associated P55IC, FAS-IC, P55DD and FASDD, or any self-associated fragment or any analogue or derivative thereof, and (c) will Any TNF-specific fusion product in (a) is mixed with any FAS-R ligand-specific fusion product in (b) to provide (after conventional isolation and purification methods) oligomeric (dimer or Higher order multimeric) receptors, which have at least the extracellular domains of both TNF-R and FAS-R, bind to each other by means of self-association of their fused IC or DD segments.

Another possibility for the preparation of the above-mentioned mixed oligomeric receptors is to co-transform appropriate host cells with the above-mentioned expression vectors, one of which encodes a TNF-specific TNF-fusion product, The other encodes a FAS-R ligand-specific FAS-R fusion product. Following expression of these various fusion products in host cells, mixed oligomeric (TNF-R/FAS-R) receptors are prepared using conventional purification and isolation techniques.

The use of these mixed affinity oligomeric receptors is primarily to neutralize TNF and FAS-R ligands, especially when both are endogenously overexpressed or reach excessive levels following exogenous administration. Recent evidence points to the possibility that there is a functional synergy between the FAS-R ligand (which is normally cell surface bound) and TNF-a (which may also be cell surface bound). Thus, in some cases, it is necessary to neutralize both ligands at the same point on the cell surface, that is, a receptor that prevents TNF from binding to it and FAS-R ligand from binding is required. Mixed affinity receptors for its receptors.

Thus, these mixed affinity receptors can be used as an active ingredient in pharmaceutical compositions for the treatment of the above mentioned conditions (see above), ie where TNF and FAS-R ligand effects are unfavorable.

Also, based on the present series of soluble oligomeric TNF-R and FAS-R and mixed TNF-R/FAS-R oligomers, it is possible to produce some soluble oligomeric receptors for other receptors, or mixtures thereof , in particular receptors for any other member of the TNF/NGF superfamily. In this case, any extracellular domain of the various receptors can be fused to the above-mentioned self-associated intracellular domain or some segment thereof, or to any other intracellular domain of a superfamily capable of self-association. district.

Any of the aforementioned recombinant proteins of the present invention can be expressed in eukaryotic cells (such as yeast, insect or mammalian cells) through appropriate expression vectors. Any relevant known technique may be used.

For example, protein-encoding DNA molecules obtained by any of the methods described above are inserted into appropriate expression vectors using techniques known in the art (see Sambrook et al., 1989). Double-stranded cDNA can be ligated into plasmid vectors by homopolymer tailing, or by restriction ligation using synthetic DNA ligase or blunt-ended ligation techniques. DNA ligase is used to join DNA molecules and undesired ligation is avoided by treatment with alkaline phosphatase.

In order to be able to express the desired protein, the expression vector should also include some specific nucleotide sequences, which contain DNA sequences with transcription and translation regulation information for the desired protein, so that many genes can be expressed. First, for a gene to be transcribed, promoters recognized by RNA polymerase must become active, and the polymerase then binds to the promoter and initiates transcription. There are a number of such promoters in use, which differ in their efficiency (strong or weak promoters). Promoters are also different in prokaryotic cells and eukaryotic cells.

Some promoters that can be used in the present invention can be constitutive, such as the int promoter of bacteriophage, the bla promoter of the β-lactamase of PBR322 and the CAT promoter of the chloramphenicol acetyltransferase gene of PPR325 It can also be inducible (inducible), such as prokaryotic cell promoters, including the main right and left promoters (PL and PR) of phage, trp , recA , lacZ , lacL , ompE and E. coli gal promoter , or trp-lac promiscuous promoter etc. (Glick, BR (1987)). In addition to the use of strong promoters to produce large amounts of mRNA, to achieve high levels of gene expression in prokaryotic cells requires the use of ribosome-binding regions to ensure efficient translation of the mRNA. One example is the Shine-Dalgarno sequence (SD sequence) appropriately positioned from the starting codon and complementary to the 16S RNA 3'-end sequence.

For eukaryotic host cells, different transcriptional and translational regulatory sequences may be used depending on the nature of the host. They may be of viral origin, such as adenovirus, bovine papilloma virus, simian virus, etc., where regulatory signals are associated with genes expressed at high levels. For example, TK promoter of herpes virus, SV40 early promoter, yeast gal4 gene promoter, etc. Considering that transcription initiates regulatory signal repression or activation, it needs to be selected so that it can regulate the expression of the gene.

The DNA molecule containing the nucleotide sequence encoding the fusion product protein of the present invention is inserted into a vector with transcriptional and translational regulatory signals that can be ligated, and these signals can integrate some required gene sequences into the host cell. Cells stably transformed by the introduced DNA can be selected by introducing one or more marker genes. The marker gene can provide nutrition to an auxotrophic host, or resistance to biocides, such as resistance to antibiotics, or heavy metals such as copper. A selectable marker gene can be directly linked to the DNA sequence to be expressed, or introduced into the same cell by co-transfection. For optimal synthesis of the proteins of the invention, several additional elements are required. These elements may include transcriptional promoters, enhancers. cDNA expression vectors having these elements include those described by Okayama, H (1983).

In a preferred embodiment, the introduced DNA molecule will be integrated into a plasmid or viral vector capable of autonomous replication in the recipient cell. Important factors in choosing a particular plasmid or viral vector include: ease, i.e., recipient cells containing the vector are easily identified and isolated from recipient cells without the vector; "Transfer" between different host cells.

Preferred prokaryotic cell vectors include such plasmids, such as plasmids capable of replicating in Escherichia coli, such as PBR322, COLE1, PSC101, PACYC184, etc. (see Maniatis et al., 1982; Sambrook et al., 1989); bacillus (Bacillus) plasmids such as PC194, PC221, PT127, etc. (Gryczan, T. (1982)); Streptomyces plasmids, including PLI101 (Kendall, K.J. et al., (1987)); Streptomyces phages, such as φC31 (Chater, K.F. et al., in: Sixth Internationalsymposium on Actinomycetales Biology (1986)), and Pseudomonas plasmids (John, J.F. et al., (1986) and Izald, K. (1978)). Preferred eukaryotic cell plasmids include BPY, vaccinia, SV40, 2-micron circle, etc., or their derivatives. These plasmids are known in the art (Botstein, D. et al., (1982); Broach, J.R. in: The Molecular Biology of the Yeast Saccharomyces: Life cycle and Inheritance (1981); Broach, J.R. (1982); Bollon, D.P. et al., (1980); Maniatis, T., in: Cell Biology: A Comprehensive Treatise vol. 3, Gene Expression, (1980); and Sambrook et al., (1989)).

Once the vector or construct-containing DNA sequence is available for expression, the DNA construct can be introduced into an appropriate host cell by any suitable method: transformation, transfection, conjugation, protoplast fusion, electroporation (electroporation), calcium phosphate precipitation, direct microinjection, etc.

The host cells used in the present invention may be prokaryotic cells or eukaryotic cells. Preferred prokaryotic hosts include bacteria such as Escherichia coli, Bacillus, Streptomyces, Pseudomonas, Salmonella typhimurium, Serratia marcescens, and the like. The most preferred prokaryotic host is E. coli. Bacterial hosts with special effects include Escherichia coli K12 strain 294 (ATCC3146), Escherichia coli x1776 (ATCC31537), Escherichia coli W3110 (F ^- , lambda ^- , prototropic (ATCC27325)), and other enterobacteria such as murine Salmonella typhi or Serratia marcescens and various Pseudomonas species. Under these conditions, the protein does not need to be glycosylated. The prokaryotic host must be able to match the regulatory sequences in the replicon (replican) and expression plasmid.

Preferred eukaryotic hosts are mammalian cells, such as cells of humans, monkeys, mice, and Chinese hamster ovary (Chinese hamster ovary, abbreviated as CHO), because they can perform post-translational modifications on proteins, including folding at the correct position ( tolding) or glycosylation. Yeast cells are also capable of post-translational modifications, including glycosylation. There are various recombinant DNA strategies that take advantage of strong promoter sequences and high copy numbers of plasmids that can be used to produce the desired protein in yeast. Yeast can recognize some leading sequences on cloned mammalian gene products and secrete peptides containing leading sequences (ie, pre-peptides, pre-peptides).

Following introduction of the vector, the host cells are grown in a selective medium that can be used to select for vector-containing cells. Expression of the cloned gene sequence results in the production of the desired protein.

Purification of the recombinant protein may be carried out by any of a number of known methods for this purpose, ie any conventional manipulation technique including extraction, precipitation, chromatography, electrophoresis and the like. Another ideal operating technique for purifying the proteins of the invention is affinity chromatography using anti-TNF receptor monoclonal antibodies produced and immobilized in a gel matrix contained within a chromatographic column. An impure preparation containing recombinant protein elutes through the column. Proteins are bound to the column by specific antibodies, and impurities are removed through the column. After washing, proteins are eluted from the gel due to changes in pH or ionic strength.

As used herein (see above) the term "Salts" refers to carboxyl salts and acid addition salts of amino acids of proteins formed by known methods. Carboxyl salts, including inorganic salts such as sodium, calcium, and salts with organic bases, such as amines, such as triethanolamine, arginine or lysine, acid addition salts include, for example, inorganic acid salts and organic salt.

"Functional derivatives" as used herein includes derivatives prepared from functional groups, by techniques known in the art, as side chains on amino acid residues, or N-terminal groups or C- The presence of side chains on the terminal group, and as long as they remain pharmaceutically acceptable, that is, they do not impair protein activity and do not confer toxicity to the drug-containing component, they are all included in the present invention, these Derivatives include fatty acid esters or amides, and N-acyl derivatives formed from amino groups and acyl moieties (such as alkanoyl or carbocyclic aroyl) or O-acyl derivatives formed from hydroxyl groups and acyl moieties.

As used herein, "portion" refers to any fragment or portion of a receptor, (either intracellular or extracellular), or a portion of a protein that binds to the intracellular region of a receptor, if the receptor retains its biological activity. live.

As mentioned above, the present invention relates to various pharmaceutical compositions, which include pharmaceutically acceptable carriers, and various active ingredients mentioned in the present invention, or their salts, functional derivatives or Mixtures of the above substances. These components can be used in any of the conditions mentioned herein, for example, conditions of oversynthesis of endogenous TNF like septic shock, cachexia, graft versus host response autoimmune diseases like rheumatoid arthritis, and the like. The mode of administration of the drug can be by any of the accepted modes of administration of similar agents and also depends on the situation to be treated, for example when used to inhibit the effects of TNF they are used as intravenous treatment in the case of septic shock , or in rheumatoid arthritis can be used as a local injection (for example, into the knee), or continuous infusion, etc. These compositions can also be used eg in cases of TNF intoxication caused by in vitro administration of excess TNF, as in the case of cancer treatment or viral disease treatment.

When administered as a drug, the pharmaceutical composition of the present invention is prepared by mixing the protein or its derivatives with pharmaceutically acceptable carriers, stabilizers and excipients, and prepared into dosage forms, such as stored in a frozen form. in the bottle. The amount of active compound administered depends on the course of administration, the disease to be treated and the condition of the patient. For example, in the inflammatory condition of rheumatoid arthritis, the amount of active compound administered by topical injection based on body weight will be less than that administered intravenously in the case of septic shock.

Other aspects of the invention will become more apparent in the following examples.

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

Example 1

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

To isolate proteins that interact with the intracellular domains of the p55 and p75 TNF receptors (p55IC and p75IC), the yeast two-hybrid system was used (Fields and Song, 1989). Briefly, the two-hybrid system is a yeast-based genetic assay to detect specific protein-protein interactions in vivo by restoring its eukaryotic transcriptional activator (activator), such as GAL4. The eukaryotic transcriptional activators, such as GAL4, have two separate domains: the DNA-binding domain and the activation domain, which, when expressed and combined to form the reconstituted GAL4 protein, can associate with the upstream activation sequence. Binding, followed by activation of promoters, regulates the expression of reporter genes, such as LacZ or HIS3 reporter genes, whose expression is easily observed in cultured cells. The coded genes of candidate proteins that interact in this system are cloned into separate expression vectors. In one expression vector, a candidate protein bias sequence was cloned simultaneously with the sequence of the GAL4 DNA-binding domain to produce a hybrid protein with the GAL4 DNA-binding domain; in another vector, the bias sequence of the second candidate protein was combined with The off-coding sequence of the GAL4-activating region was cloned simultaneously to generate a hybrid protein with the GAL4-activating region. The two hybrid vectors were co-transformed into yeast host cell lines containing the lacZ or HIS3 reporter gene. This reporter gene is regulated by an upstream GAL4 binding site. Only those transformed host cells expressing two hybrid proteins that interact with each other (co-transformants) were able to express the reporter gene. In the presence of the lacZ reporter gene, host cells expressing this gene will turn blue when X-gal is added to the culture. Thus, the blue clones indicate the fact that the candidate proteins of the two clones are able to interact with each other.

Using this two-hybrid system, clone the intracellular region at ρ55IC and ρ75IC into the vector ρGBT9 (carrying the GAL4 DNA-binding sequence, provided by CLONTECH, USA, see below) to generate a fusion protein with the GAL4 DNA-binding region (Similarly, a fragment of the intracellular region FAS-IC and 55IC, ie, 55DD, was cloned into pGBT9 to isolate other IC-binding proteins, see Example 3 below). To clone p55IC and p75IC into pGBT9, clones of the full-length cDNA sequences encoding p55TNF-R (Schall et al., 1990) and p75TNF-R (Smith et al., 1990) were used, wherein the intracellular region (IC ) was cleaved as follows: the ρ55IIC coding fragment was excised with EcoRI and SalI enzymes, and the EcoRI-SalI fragment containing the ρ55IC coding sequence was isolated according to conventional methods, and embedded in the pGBT9 vector, which was composed of EcoRI and SalI in its The multiple cloning site (MCS) was cut; the p75IC coding fragment was cut out with BspHI and SalI enzymes, and the BspHI-SalI fragment containing the p75IC coding sequence was isolated according to conventional methods, and Klenow enzyme was added to generate a fragment that could be inserted into the pGBT9 vector. The vector is also cut by Smal and SalI.

Then the above-mentioned hybrid (chimeric) vector and the PGADGH vector with GAL4 activation region were co-transfected into the HF7C yeast host cell line, (respectively, one was co-transfected with the P55IC hybrid vector, and the other was co-transfected with the P75IC hybrid vector) The PGADGH vector also contains a cDNA library from human Hela cells (all of the above vectors, ρGBT9 and ρGAD GH carrying a Hela cell cDNA library, and the yeast line as part of the MATCHMAKER two-hybrid system, #PT1265-1 are from Clontech Laboratories, Inc., USA .purchased). Co-transfected yeast were selected for their ability to grow in media lacking histidine (His ^- medium), ie growing clones that appeared to be positive transformants. Then test the ability of the selected yeast clones to express the lacZ gene, that is, their LACZ activity, which is carried out by adding X-gal to the medium, and the β-galactosidase encoded by the lacZ gene degrades X-gal to produce blue color product. Thus, blue colonies indicate an activated lacZ gene. The GAL4 transcriptional activator is present in an activated form in transformed clones and is necessary for the activation of the lacZ gene, i.e., the GAL4 DNA-binding region encoded by one of the preceding hybrid vectors is appropriately combined with the GAL4 activating region encoded by the other hybrid vector Binding, is necessary for the activation of the Lac gene. This association is only possible if the two proteins fused to each GAL4 region are able to stably interact (bind). Thus, isolated His+ and blue (LACZ+) colonies were colonies: they were co-transfected with a vector encoding p55IC and a vector encoding a protein product from human Hela cells that could stably bind to p55IC; or were co-transfected with a vector encoding p75IC The vector was co-transfected with a vector encoding a protein product from human HeLa cells that stably binds to p75IC.

Using conventional methods, isolate the plasmid DNA from the aforementioned His+, LACZ+ yeast clones, and electroporate them into E. coli strain HB101, and then select Leu+ and ampicillin-resistant transformants, which carry AmpR and Leu2-containing Hybrid rhoGAD GH carrier of two biased sequences. Therefore, these transformants were clones carrying the sequence encoding the protein newly determined to bind p55IC or p75IC. Plasmid DNA was then isolated from these transformed E. coli and retested as follows:

(a) It was retransformed into yeast strain HF7 with the original intracellular domain hybrid plasmid (hybrid pGTB9 carrying the p55IC or p75IC sequence) as previously described. As a control, vectors carrying unrelated protein coding sequences such as pACT-lamin, or pGBT9 alone were used for co-transformation with plasmids encoding p55IC-binding protein or p75IC-binding protein. The co-transformed yeasts were then tested for growth in His ^- medium alone or in media containing different concentrations of 3-aminotriazole at the same time.

(b) Co-transform yeast host cells of the SFY526 line with the plasmid DNA mentioned in (a), the hybrid plasmid of the original intracellular region, and the control plasmid, and test its LACZ+ activity (the efficiency of β-gal formation, that is, blue color production).

The above test results show that: when measured by the color of the colony, the growth pattern of the colony in the His ^- medium is fully consistent with the pattern of LACZ activity, that is, His+ clones are also LACZ+. In addition, the LACZ activity in liquid (under ideal culture conditions) culture was measured after the hybrid plasmid of the GAL4 DNA binding region and the activation region was transfected into the SFY526 yeast host. The SFY526 yeast host had better GAL4 transcription than the HF-7 yeast host cell Inducibility of activators to LACZ.

The results of the co-transfection described above are presented in Table 1 below, from which it is evident that many proteins were found to be able to bind to p55IC or p75IC, namely: the protein called 55.11 was able to bind to p55IC, and the proteins called 75.3 and 75.16 were able to bind to p55IC. Binds to ρ75IC. All these ρ55-IC- and ρ75IC-binding proteins are real human proteins, and they are all encoded by the cDNA sequence in the cDNA library of human Hela cells. GAL4 activation-domain sequence fusion.

Interestingly, it was also found that some fragments of p55IC themselves, proteins called 55.1 and 55.3, were able to bind p55IC. This will also be discussed in Example 2.

Table 1 Summary of characteristics of some cDNA clones isolated from the two-hybrid system

DNA-binding Activation-zone Colony color In liquid culture samples district bastard bastard LacZ activity pGBT9-IC55 --- white 0.00 pGBT9-IC55 55.1 orchid 0.65 pGBT9-IC55 55.3 orchid 0.04 --- 55.1 white 0.00 --- 55.3 white 0.00 pACT-Lamin 55.1 white 0.00 pACT-Lamin 55.3 white 0.00 pGBT9 55.1 white 0.00 pGBT9 55.3 white 0.00 pGBT9-IC55 55.11 orchid ND --- 55.11 white ND pACT-Lamin 55.11 white ND pGBT9 55.11 white ND pGBT9-IC75 75.3 orchid ND pGBT9-IC75 --- white ND --- 75.3 white ND pACT-Lamin 75.3 white ND pGBT9 75.3 white ND pGBT9-IC75 75.16 orchid ND --- 75.16 white ND pACT-Lamin 75.16 white ND pGBT9 75.16 white ND

In Table 1 above, plasmids and hybrids encoding the GAL4 DNA-binding region and the GAL4 activation region are listed as follows:

DNA-binding region hybrids:

ρGBT9-IC55: the full-length intracellular domain of ρ55-TNF-R (ρ55IC)

ρACT-Lamin: irrelevant protein-lamin

ρGBT9: the carrier itself

ρGBT9-IC75: the full-length intracellular region of ρ75-TNF-R (ρ75IC) activation region hybrid:

55.1 and 55.3 correspond to fragments of the intracellular region of p55-TTNF-R

55.11 is a new protein related to ρ55-TNF-R

75.3 and 75.16 are novel proteins related to ρ75-TNF-R

The cDNAs of the above clones encoding the following proteins: 55.11, 75.3 and 76.16 of the novel p55IC- and p75IC-binding proteins were sequenced by conventional DNA sequencing methods. Partial sequences of all these protein coding sequences are listed in Figure 1a-c, wherein Figure 1(a) lists the sequence cDNA encoding protein 55.11, and Figure 1(b) lists the partial sequence of cDNA encoding protein 75.3, Figure 1 (c) Partial sequence of cDNA encoding protein 75.16 is listed. In Figure 1(d), the amino acid sequence of protein 55.11 deduced from the nucleotide sequence of Figure 1(a) is shown.

However, it should be mentioned that the partial sequence of the cDNA encoding the 55.11 protein has also been reported by Khan et al. (1992) in the study of the cDNA sequence of the human brain. Brain cANA sequencing and mapping its physical and genetic maps. However, Khan et al. did not provide information on any other characteristics and functions of the protein encoded by the cDNA sequence of 55.11, and these functions and other analyzes were not the purpose of the study by Khan et al.

55.11 Protein analysis and identification

(a) Methods and materials

(i) Cloning of cDNA of 55.11

According to the analysis of the cDNA of the 55.11 protein: (for example, see the following Northern blot analysis), the above-mentioned 55.11 cDNA cloned by the two-hybrid screening method only represents a partial sequence of the 55.11 cDNA, including nucleotides 925-2863 The fragment between (see Figure 1(a)), the nucleotide sequence encodes a fragment between amino acids 309-900 (see Figure 1(d)). 55. The rest of the 11cDNA (the segment between nucleotides 1-924 (Figure 1(a)) encodes the segment between amino acids 1-308 (Figure 1(d)), which is obtained by conventional methods, that is, by PCR technology Cloned from a human fetal liver cell cDNA library.

The full-length nucleotide sequence of 55.11 (Figure 1(a)) was determined from two aspects by the dideoxy chain termination method

(ii) β-galactosidase expression test of double hybrid line

The β-galactosidase expression assay was performed as described above. But in some experiments, PVP16 vector (containing VP16 active region) was used instead of PGAD-GH (Gal4 active region) carrier. The numbering of amino acid residues in the protein encoded by the cDNA insert refers to the Swiss-Prot database. Deletion mutants were obtained by PCR, and point mutations were obtained by oligonucleotide-directed mutagenesis (Kunkel, (1994).

(iii) Northern Analysis

Using conventional techniques, the total RNA separated by TRI kit (product of American Center for Molecular Research, Cincinnari Oh., U.S.A) was denatured in formaldehyde/formamide buffer, electrophoresed on agarose/formaldehyde gel, and then Blot in 10×SSPE buffer to Gene ScreenPlus membrane (product of DuPont, USA). The blot was hybridized with a part of cDNA at 55.11 (see above, the segment between nucleotides 925-2863), and was labeled with a random primer kit (product of Mannheim Biochemical Company, Boehringer, Germany), and after strict washing , autoradiography for one week.

(iv) Expression of 55.11 cDNA in Hela cells and binding of 55.11 protein to glutathione sulfur transferase fusion protein of p55IC

The fusion protein of glutathione sulfur transferase (GST) and ρ55IC (GSF-ρ55IC) and the fusion protein of glutathione-S-transferase and ρ55IC excised below the 345th amino acid sequence (GST-ρ55IC345) were synthesized and Adsorb to glutathione-sepharose beads as described in Example 2 below. (cf. Smith and Corcoran, 1994); Frangioni and Neel, 1993). The cDNA of 55.11 (1-2863 nucleotides is the full-length cDNA of 55.11), the cDMA of FLAG-55.11 and the cDNA of luciferase were expressed in Hela cells. FLAG-55.11 is the coding segment of 309-900 amino acid residues in 55.11 protein, (55.11 partial cDNA (nucleotides 925-2863) cloned by double hybrid screening line) N-terminal is connected with FLAG octapeptide. The expression of the fusion protein in Hela cells is realized by the expression vector (HtTA-1) regulated by tetracycline. Such Hela cells express tetracycline-regulated transactivators (see Example 2 below, Gossen and Bujard, 1992). The expressed protein was radioactively labeled with ( ³⁵ S) methionine ( ³⁵ S) cysteine (products of DuPont Company of the United States and Amer-sham Company of the United Kingdom), followed by lysis of Hela cells, followed by immunoprecipitation, and then Conjugation of the marker protein to the glutathione thiotransferase fusion protein was carried out as described below (Example 2), but 0.5% was used instead of 0.1% NonidetP-4 in the cell lysis buffer. Use the immune antiserum (1:500 dilution) against the 55.11 glutathione sulfur transferase fusion protein containing the amino acid 309-900 segment, and the anti-FLAG octapeptide mouse monoclonal antibody to obtain 55.11 and FLAG-55.11 immunization Precipitate ( _M2 ; Eastman Kodak; 5 μg/ml cell lysate).

(b) Binding of 55.11 protein to ρ55IC in transformed yeast

This study was to clarify the binding properties of 55.11 and ρ55IC, especially the binding-related segments of the two proteins. The "DNA binding region" construct was used as a "bait" to bind the "prey", that is, the amino acid sequence of the part of the 55.11 protein encoded in the construct (residues 309-900, as originally isolated) to be fused to the GAL4AD and VP16AD vectors the "active area". Furthermore, various deletion mutants of 55.11 were constructed and fused with the "active region" of the GAC4AD vector. (eg 55.11 mutant with only 309-680 and 457-900 amino acid fragments). Binding of various "binding domain" constructs to various "active domain" constructs was tested in transfected SFY526 yeast cells. And through the two-hybrid β-galactosidase expression filter membrane binding test, the binding was determined. Non-related proteins SNF1 and SNF4 were used as positive controls for the "binding region" and "active region" constructs, respectively; Gal4 (PGAD-GH) and VP16 (PVP16) empty vectors were used as negative controls for the "active region" constructs, and Gal4 (PGBT9 ) empty vector as a negative control for the "binding region" construct. The results of the measurement are shown in Table 2 below. The symbols "+++" and "++" in the table represent the color change (positive binding result) within 20-60 minutes after the start of the measurement respectively; No color develops within hours (negative result). Blank places in the table indicate undetermined binding assays.

Table 2

Binding of 55.11 protein to P55IC in transformed yeast

From the results listed in Table 2 above, it can be inferred that 55.11 binds to a certain position of P55IC, which is different from the "death domain" (328-426 amino acid residues) of P55IC

The 55.11 protein bound more efficiently to the truncated P55IC protein lacking the lethal region (206-328 constructs in Table 2) than to the non-truncated P55IC.

55.11 was also able to bind to protein constructs further truncated at the C-terminus (constructs 206-308) and to constructs in which the death domain and proximal membrane region were deleted (constructs 243-328). However, the 55.11 protein was not involved in the binding of constructs with amino-terminal deletions up to amino acid 266 (see Table 2). These findings indicated that the binding position of 55.11 is located in the amino acid region between 243-308 of P55IC, and the N-terminus of the binding position is in the amino acid region between 243-266.

Transfer of 55.11 cDNA from a clone-derived 'trap' construct containing the Gal4 active region to a capture construct containing the VP16 active region did not reduce the binding effectiveness of the 55.11 protein to P55IC (Table 2) . Thus, structures involved in binding appear to be located within the 55.11 molecule, but do not appear to include the fusion site of 55.11 to the active domain.

However, binding of 55.11 to the P55IC protein can be prevented by restricted excision of the C-terminus (55.11 construct 309-680) or N-terminus (55.11 construct 457-900) of the 55.11 protein. (The 309 amino acid residues are the first amino acid residues of the 55.11 protein encoded by the partial cDNA screened out by the two-hybrid method).

The observed binding of 55.11 to p55IC appears to be specific. Because 55.11 does not bind to other proteins, including three receptor proteins of the tumor necrosis factor/nerve growth factor receptor family (P75-R, Fas/AP01 and CD40) and other proteins such as lamins and cyclins D (data not published). It is worth emphasizing that of the other TNF/NGF receptor proteins tested, fragments containing intracellular domains were also tested, namely human FAS-R (amino acid residues 175-319), CD40 (amino acid residues 216-277) and P75-TNF-R (amino acid residues 287-461), neither of which binds to 55.11 (unpublished data).

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

The test cell lines include Hela cell line, CEM cell line, Jurkat cell line and HepG2 cell line, which are derived from human epithelial cancer cells, acute lymphoblastic T-cell leukemia, acute T-cell leukemia and liver cancer cells, respectively. The initially isolated cDNA (nucleotides 925-2863) was used as a probe and the sample contained 10 μg RNA/lane. The results of the Northern analysis are shown in Figure 2, where the Northern blot is reproduced.

It can be seen from Figure 2 that Northern analysis was performed in several cell lines with 55.11cDNA as a probe. A single hybrid transcript of approximately 3 kB was found, larger than the 55.11 cDNA (3 kB) originally isolated. Using oligonucleotide primers corresponding to the 55.11 sequence, the 5' extended sequence with a length of about 1 kB was cloned by PCR (polymerase chain reaction), and the length of the 5' extended sequence plus the length of the initially cloned cDNA was approximately Transcript length at 55.11. The 3kB cDNA includes the above two parts (1kB sequence and 2kB sequence), which can be efficiently expressed in transfected Hela cells (see below) and produce a protein of about 84kDa. This indicates that the 3kB cDNA contains a translation initiation site.

(d) In vitro binding of 55.11 protein to glutathione thiotransferase fusion protein containing the P55IC fragment.

In order to confirm that 55.11 can indeed bind to P55IC, and exclude yeast protein from participating in this process, the fusion protein of glutathione sulfur-transferase P55IC produced by bacteria and transfected HeLa cells was detected, which was produced by 3kB 55.11cDNA (55.11 full-length ) in vitro interactions between encoded proteins. In this study, cDNAs encoding full-length 55.11, FLAG-55.11 (amino acid residues 309-600 of 55.11 encoded by the initially cloned partial cDNA fused to the N-terminus of the FLAG8 peptide) and luciferase (as a control) were all transfected. expressed in transfected HeLa cells and radiolabeled with [ ³⁵ S]methionine and [ ³⁵ S]cysteine by metabolic methods. The following proteins are fused to GST, the full-length PP55IC (glutathione-P55IC fragment) and the P55IC fragment (glutathione P55IC345), which ends at 345 amino acid residues from the C-terminus of P55IC and removes most of the "death domain" ” formation (see Table 2). Glutathione alone served as a control. Antibody against 55.11 protein was used to immunoprecipitate lysates of transfected cells when full-length 55.11 protein was used to bind glutathione fusion protein, or when FLAG-55.11 fusion protein was used to bind glutathione fusion protein , an antibody against the FLAG8 peptide was used to immunoprecipitate lysates of transfected cells. Protein analysis was performed by SDS polyacrylamide gel electrophoresis (SDS-PAGE, 10% acrylamide) followed by autoradiography.

Figures 3A and B show the results of the above SDS-PAGE gel autoradiography. Figure 3A shows the binding of full-length 55.11 protein (55.11-full length) to various glutathione fusion proteins, and Figure 3B shows the binding of Flag-55.11 fusion products to various glutathione fusion proteins. The rightmost lane in Figure 3A shows the results of immunoprecipitation of control cell lysates transfected with full-length 55.11 only and immunoprecipitated with anti-55.11 antibodies (α55.11 Abs). The rightmost lane in Figure 3B shows the results of immunoprecipitation of control cell lysates transfected with FLAG-55.11 only and immunoprecipitated with anti-FLAG antibodies (αFLAG-Abs).

Thus, it is apparent from the results in Figure 3A and B that the protein encoded by the full-length 55.11 cDNA can be expressed in HeLa cells and can be combined with the truncated P55IC (glutathione-P55IC) containing all or losing most of the lethal region. A fusion protein of P55IC (glutathione-P55IC345) was associated (Fig. 3A). The full length 55.11 protein does not bind glutathione itself (control). Similarly, the protein encoded by the partially cloned 55.11 cDNA expressed in HeLa cells can form a fusion protein (FLAG-55.11) with the FLAG8 peptide, which can combine with glutathione-P55IC and glutathione in vitro -P55IC345 bound, but not glutathione itself (Fig. 3B). The above results thus provide additional (see (b) above) evidence that 55.11 binds to a region upstream of the P55IC "death domain", ie a region close to the P55IC transmembrane region.

In addition, the above studies also show that according to the results of the present invention, an anti-55.11 antibody has been successfully synthesized ( FIG. 3A ).

(e) Comparison of deduced amino acid sequences of human 55.11 protein and related proteins in lower organisms and sequence characteristics of 55.11 protein

As mentioned above in the present invention, the full-length 55.11 cDNA has been cloned and sequenced (see the nucleotide sequence in Figure 1 (a)) and the entire amino acid sequence of 55.11 deduced from the cDNA sequence (see Figure 1 (d) amino acid sequence). A search of the database (Gene Bank TM/EMBL Data Bank) showed that the human 55.11 cDNA partial sequence (accession numbers T03659, Z19559 and F091287) and the mouse counterparts (accession numbers X80422 and Z311477) had been determined in the random sequencing of the cDNA library. The cDNA coding sequence of a 596-amino acid human protein present in human liver cancer HC10 cell cultures is similar to the 55.11 cDNA coding sequence. However, the liver cancer protein lacks the N-terminal fragment (amino acid 1-297) corresponding to 55.11, and is also different from 55.11 in the 297-377 and 648-668 amino acid segments corresponding to 55.11. Database search also showed that there are also proteins with high sequence homology with 55.11 in yeast (Saccharomyces) plants, (Arabidopsisthaliana), and worms (Caenorhabditis elegans). The 55.11 protein appears to have an evolutionarily conserved function. In yeast, two yeast proteins (open reading frames YHR027C and SEN3) are known to have DNA sequences similar to 55.11 and similar in size to 55.11. YHR027C is only known through sequencing of gene clones, whereas SEN3 has been cloned as a cDNA molecule. The site similar to SEN3 in 55.11 was also associated with its similar site in YHR027C. Although evidence suggests that 55.11 is more similar to YHR027C than it is to SEN3. Information on the DNA sequences obtained from the plant (Arabidopsis thaliana) and worm (Caenorhabditis legans) proteins, albeit only partial sequences, clearly shows that these proteins share a pair 55.11 similarity to the YHR027C yeast protein. So far, only yeast SEN3 has been elucidated among the four proteins, and its homology with 55.11 has been limited. SEN3 was identified as the counterpart of the P112 subunit of the 20S proteasome activator in yeast (the 20S proteasome is the hydrolytic core of the 26S proteasome) [Rechsteiner et al., 1993. De Martino et al., 1994] (M.R.Culbertson and M.Hock strasser, private communication).

Figure 4 schematically shows a comparison of the deduced amino acid sequence of the human 55.11 protein with the aforementioned related proteins present in lower organisms. The amino acid sequence compared in Fig. 4 is encoded by the following cDNA: 55.11cDNA (see Fig. 1(d)) (SEQ ID NO: 14); an open reading frame (YAR027C), present in the eighth chromosome of yeast (Saocharomyces cerevisiae) In a cosmid; (nucleotides 21253-24234, Accession No. U10399) (SEQ ID NO: 15); SEN3 cDNA encoding a yeast protein (Accession No. L06321) (SEQ ID NO: 16); part of a plant (Arabidopsis thaliana) protein cDNA (Accession No. T21500) (SEQ ID NO: 17); and a partial cDNA of a worm (Caenorhabditiselegans) protein (Accession No. D27396) (SEQ ID NO: 18). The "KEKE" sequence of 55.11 is indicated by a solid line, and the AYAGS(x)8LL sequence is indicated by a dashed line. Sequences were aligned using the PILEUP and PRETTYBOX programs of GCG software. The spacing used to increase the alignment of lines is indicated by dashes. Various sequence features or motifs in the human 55.11 amino acid sequence are described below: Except for a "KEKE" repeat sequence ranging from lys614 to Glu632 (bottom black line in Figure 4), no 55.11-encoded protein was found Contains conserved amino acid sequence motifs. This "KEKE" sequence is present in many proteins, including proteasome subunits and molecular proteins. The "KEKE" sequence can promote the association of protein complexes (Realini et al, 1994), and the AYAGS(X) ₈ LL sequence appears twice in the 55.11 protein (see Figure 4 for positions 479 and 590); The significance of the role has not been described.

(f) Sequence characteristics of the region associated with 55.11 protein binding in P55IC

As described above, (see (b) and (d), the 55.11 protein binds to the region between amino acids 243-308 of P55IC (the N-terminus of the binding site is between amino acid residues 243-266) This region is upstream of the "death domain" and is closer to the transmembrane region of P55-TNT-R. In the P55IC region where 55.11 binds, it is rich in proline, serine and threonine residues. However, this region does not contain the RPM1 and RPM2 proline-rich motifs that occur in several other cytokine receptors (Oneal and Yulee, 1993). In the segment between amino acid residues 243-266 , (deletion of this segment can prevent P55-R from binding to 55.11), (see (b) and (d) and Table 2), the structure of two serines and two threonines followed by proline residues can Potential site for phosphorylation by MAP kinase, CDC2 and other proline-dependent kinases (Seger and Krebs, 1995). Phosphorylation of this site in the receptor protein may affect its binding to 55.11 protein .

In view of the above findings on protein 55.11 and its binding to P55IC, it can be concluded that, according to the present invention, a new protein has been found that binds to a specific region upstream of the "death domain" of P55IC, which binding can Affects TNF-mediated activity without causing cell death. The 55.11 binding domain has previously been shown to be involved in the induction of nitric oxide synthase (Tartaglia et al, 1993) and also appears to be involved in the activation of sphingomyelinase by tumor necrosis factor. (Wiegmam et al, 1994). Thus, 55.11 binding to the P55-TNF-R intracellular region (P55IC) may affect or be involved in the following processes: (i) providing signals for the above-mentioned or other TNF effects; (ii) protein folding or processing (as described by 55.11 similarity to a 26S proteasome subunit); (iii) regulation or expression of P55-TNF-R activity.

Example 2

Self-association ability, ability to cause cell death and others of P55TNF receptor intracellular domain (P55IC) Characteristics and activities and the intracellular domain of the associated Fas/AP01 receptor

As described in Example 1 above, it was found that the intracellular domain of P55-TNF-R (P55IC) has the ability to self-associate, and also fragments of P55IC, protein 55.11 and protein 55.3, were found to bind to P55IC.

Binding of TNF to P55-TNF-R is known to result in a cytocidal effect on cells bearing this receptor. Moreover, antibodies raised against the extracellular domain of the receptor can themselves trigger this effect, which is related to the effectiveness of antibody cross-linking to the receptor.

Furthermore, mutational studies [Tartaglia et al (1993); Brakebusch et al., (1992)] indicated that the function of P55-R depends on the integrity of its intracellular domain. Therefore, it is also indicated that the signal initiation of the cytocidal effect of TNF is generated as a result of the self-association of two or more intracellular domains of P55-R (P55IC), and this self-association is caused by the aggregation of receptors. of. According to the present invention, these results provide further evidence for this insight. These results suggest that intracellular expression of the intracellular domain of P55-R triggers cell death in the absence of a transmembrane or intracellular domain. These free P55-R intracellular domains exhibit the ability to self-associate, which may also account for their TNF-independent ability to act. The fact that full-length p55-R signaling is dependent on TNF stimulation suggests a reflexive activity of the receptor's transmembrane or extracellular domain that reduces or prevents self-association.

The self-association ability of the intracellular domain (P55IC) of P55-R was discovered quite by accident during an attempt to clone effector proteins that interact with this receptor (see Example 1 above). For this purpose we applied the "two-hybrid" technique described above. In addition to the discovery of the new protein 55.11 that can bind to P55IC, three other cloned HeLa cell cDNAs containing cDNA sequences encoding part of the intracellular region of P55-R were also found, suggesting that P55IC is capable of self-association. Two of these clones were identical and contained an insert encoding amino acids 328-426 (designated clone 55.1) encoding protein fragment 55.1 of P55IC, and the third contained a longer insert encoding amino acids 277-426. (Denoted as clone 55.3, encoding protein fragment 55.3 of P55IC).

Furthermore, we analyzed the interaction of two chimeric proteins of p55IC produced by bacteria in vitro. One of the proteins is fused to maltose-binding protein (MBP), and the other chimeric protein is fused to glutathione-sulfur transferase (GST). These chimeric proteins were constructed, cloned and expressed using conventional methods. After their expression, the self-interaction of the P55-R intracellular region (P55IC) was determined by measuring the interaction of the bacterially produced chimeric proteins GST-IC55 (Mr-51kD) and MBP-IC55 (Mr-67kD) as described above . An equal amount of GST-IC55 chimeric protein (sample lanes 1-4 in Figure 5) or GST alone (sample lanes 5-8 in Figure 5) was bound to glutathione-agarose beads (Sigma), and then mixed with Equal amounts of MBP-IC55 fusion protein were incubated in one of the following buffers:

(i) Buffer I (20 mM Tris-HCl, pH 7.5, 100 mM KCl, 2 mM CaCl ₂ , 2 mM MgCl ₂ , 5 mM DTT, 0.2% Triton X100, 0.5 mM PMSF, 5% glycerol). The samples in lane 1 and lane 5 in Figure 5 were treated with this buffer.

(ii) Buffer I containing 5 mM EDTA instead of _MgCl2 . Samples in lanes 2 and 6 in Figure 5 were treated with this buffer.

(iii) Buffer I containing 250 mM KCl instead of 100 mM KCl, the samples in lanes 3 and 7 in Figure 5 were treated with such a buffer.

(iv) Buffer I contained 400 mM KCl instead of 100 mM KCl, and samples in lanes 4 and 6 in Figure 5 were treated with such a buffer.

After incubation with rotation at 4°C for 2 hours, the agarose beads were washed with the same buffer, followed by boiling in SDS-PAGE buffer, and then electrophoresed by PAGE. The proteins on the gel were subsequently blotted onto a nitrocellulose membrane and stained with a polyclonal antiserum against MBP. Figure 5 shows the results of this stained western blot analysis, the samples in lanes 1-8 are as described previously.

From Figure 5, it can be clearly observed that the binding of P55IC-MBP chimeric protein to P55IC-GST chimeric protein (lanes 1-4) has nothing to do with divalent cations, and is not affected by high concentrations of salt ions (0.4MKCl) . It can be concluded that P55IC has self-association ability.

To assess the functional significance of p55IC self-association, we attempted to express p55IC in the cytoplasm of cells sensitive to the cytocidal effects of TNF. Considering that p55IC may transform or be toxic to cells, we chose to express it in an inducible way. That is, using the recently developed mammalian expression system regulated by the tightly regulated tetracycline (Gossen and Boujard, 1992). Expression of p55IC caused massive cell death (Fig. 6, right column). Dying cells exhibited the same cell surface leakiness observed during TNF-killed cells. The P55IC construct transfected cells, in the presence of tetracycline, the expression of the HD10-3-regulated construct was reduced by 10 ⁵ times, but still caused the death of some cells. Although it was significantly lower than that observed in the absence of tetracycline. (Fig. 6, left). As a control, cells transfected with a control construct containing luciferase cDNA showed no signs of death (results not published).

In the absence of receptor expression in the transmembrane or extracellular domains, p55IC can trigger cell death. This provides further evidence that this region is involved in signaling initiation. Furthermore, it was shown that no other part of the receptor protein plays a direct role in the initiation of this signal. Studies of the effect of mutations on the function of the p55IC, including those studied in the invention, indicate that the amino acid segment located between residues 326-407 of all amino acids is most critical for the function of the p55IC. This region shows significant sequence similarity to the intracellular regions of two other receptor proteins. These two receptors are the Fas receptor and CD40 receptor, which are evolutionarily related to P55TNF-R, in which the Fas receptor can trigger cell death signals (Itoh et al, 1991; Oehm et al, 1992) and the CD40 receptor can Promotes cell growth (Stamenkovic et al, 1989). Therefore, the sequence of this region seems to constitute a conserved motif and play some important role in signal initiation. Since it does not resemble known motifs of enzymatic activity, it appears to signal in an indirect manner. That is, it may be a docking site for a signaling enzyme or a docking site for a protein that transmits a stimulatory signal to a signaling enzyme. P55IC, Fas receptor and CD40 can all be stimulated by antibodies raised against their extracellular domains. It was also shown that this stimulation is related to the ability of the antibody to cross-link to the receptor. Thus, it appears that the initiation of the signal is the result of the interaction of two or more intracellular domains due to aggregation of the extracellular domain. This interaction has also been shown to be associated with the initiation of receptor signaling in some studies (Brakebusch et al, 1992), which showed that receptors are rendered nonfunctional due to mutations in the intracellular domain, i.e., the normal receptors for which they are expressed The function of has a "dominant negative" effect. Aggregation of the TNF response by p55IR is thought to occur in a passive manner, but because each TNF molecule in the form of a homotrimer binds two or three receptor protein molecules. However, the present findings suggest that the process occurs slightly differently.

The self-association propensity of the p55IC suggests that this region plays an activating role in its aggregation induction. Furthermore, activation of p55IC appears to be sufficient to initiate signaling because, while independent of the expression of other receptor molecules, p55IC can trigger cell death in the absence of TNF or other external stimuli. But when it is expressed as a full-length receptor, p55-TNF-R cannot signal unless stimulated by TNF. So it was hypothesized that when P55-TNF-R is activated, TNF actually overcomes some inhibitory mechanism that prevents the self-association of the intracellular domain, and that the inhibitory effect is due to the association of P55IC with the rest of the receptor protein. This inhibition may arise due to orientation effects exerted by the transmembrane and extracellular domains on the intracellular domain, or due to the association of some other protein with the receptor, or may simply be due to the receptor being localized on the plasma membrane. The amount of body protein is limited. It is worth noting that this regulatory mechanism is quite efficient, and it is estimated that the binding of only one TNF molecule to a cell is sufficient to trigger the death of the cell.

Spontaneous signaling independent of ligands can lead to widespread perturbation of the processes regulated by the receptor. The best known example is the deregulation of growth factor receptors. Initiation signals that arise spontaneously due to mutations, such as those that cause spontaneous aggregation of receptors, play an important role in the uncontrolled growth of tumor cells. It is well known that excessive induction of TNF effects is the cause of many diseases. Free intracellular domains of P55-TNF-R (P55ICs) may contribute to this excessive effect, independent of TNF signaling. It seems possible that the cytopathic effects of certain viruses and other pathogens do not arise from their direct cytocidal effects but from the proteolysis of the intracellular domain of P55-TNF-R, leading to Cytocidal effect similar to TNF.

In order to further elucidate the segment related to self-association ability and ligand-independent cytotoxicity in P55IC, and also to detect whether other related members in the TNF/NGF receptor family (such as FAS-R) also have self-association ability The combined intracellular domain and ligand-independent effects are also studied in detail below.

(a) General methods and materials

(i) Double hybrid screening and detection of β-galactosidase expression in double hybrids

Some cDNA inserts were cloned by PCR from full-length cDNA previously cloned in our laboratory or from purchased cDNA libraries encoding P55-IC and its mutants, Fas-IC and Other proteins (see Table 3). Yeast (SFY526 reporter line (Bartel et al., 1993)) was transformed with the PGBT-9 and PGAD-GH vectors containing the above cDNA (containing the DNA-binding region (DBD) and active region (AD), respectively), in which the β-galactosidase The expression of the protein was determined by the liquid assay (Guarente, 1983), and it could also be determined by the filter binding assay, both of which produced essentially the same results (unpublished).Using the HF7c yeast reporter line, according to the manufacturer's recommendation, purchased from Gal4AD-marked Hela cell cDNA library (Clontech, Palo Alto, Ca., USA) was screened for proteins that could bind to the intracellular region of P55-R (P55-IC) by two-hybrid screening (Fields and Song, 1989). The positivity of the isolated clones was determined by (a) proton transfer of the transformed yeast to histidine when grown in the presence of 5 mM 3-aminotriazole, (b) expression of β-galactosidase, (c) Specificity test (interaction with SNF ₄ and lamin fused to Gal4DBD).

(ii) In vitro self-association of bacterially synthesized P55-IC fusion protein

The synthesis of glutathione S-transferase (GST) and glutathione S-transferase-P55-IC fusion protein (GST-P55-IC) was carried out according to the method described in the literature (Frangioni and Neel, 1993; Ausubel et al. , 1994). Maltose binding protein (MBP) fusion protein was obtained through PMalcRI vector (New England Biolabs) and purified on amylose resin column. The study of the interaction between MBPP and GST fusion protein was carried out by incubating glutathione-agarose beads with GST and MBPP fusion protein in turn (5 μg protein/20 μl beads, the first incubation was 15 minutes, the second incubation was 2 hours, all at 4°C). The incubation with the MBP fusion protein was carried out in the following buffer, that is, it contained: 20mM Tris-HCL, pH7.5, 100mM Kcl, 2mM Cacl ₂ , 2mM MgCl ₂ , 5mM dithiothreitol (DDT), 0.2 % Triton x 100, 0.5 mM benzylthiofluoro, 5% (v/v) glycerol, or where indicated, in the above buffer containing 0.4 M KCl or 0.5 mM EDTA instead of MgCl ₂ . The association of MBP fusion protein was analyzed by SDS polyacrylamide gel (10% acrylamide) electrophoresis, and then analyzed by Western blotting. Anti-MBP mouse antiserum (synthesized in our laboratory) and goat anti-rabbit immunoglobulin linked to root peroxidase were used as probes in the above blot analysis.

(iii) Induced expression of P55-R and its fragments in Hela cells

Hela cells expressing tetracycline-regulated transactivator factors were developed by Gossen and Bujard (the HtTA-1 clone (Gossen Bujard, 1992)), and the cells were grown on Dulbecco's modified Eagle's medium, which contained 10% fetal bovine serum, 100 μg/ml penicillin, 100 μg/ml streptomycin and 0.5 mg/ml neomycin medium. The cDNA insert encoding P55-R or a fragment thereof was transformed into a tetracycline-regulated expression vector (pUHD10-3, kindly provided by H. Bujard). Cells were then transfected with the above expression vector (5 μg DNA/6 cm plate) by calcium phosphate precipitation method (Ausubel et al. 1994). The effect on the transient expression of transfected proteins was determined at an intra-finger time after transfection in the presence or absence of tetracycline (1 μg/ml). Cells were transfected with human P55-IC cDNA in the pUHD10-3 vector, and stably transfected cell clones were established by transfecting the cDNA with a hygromycin-resistant plasmid into HtTA-1 cells in the presence of tetracycline, And use hygromycin 200μg/ml to screen out resistant clones. cDNA expression was obtained after removal of the tetracycline. In general, tetracycline will always be present in the cell growth medium.

(iv) Determination of TNF-like effects triggered by inducible expression of P55-R and its fragments

The effect of induced receptor and TNF expression on cell viability was determined by the neutral red uptake method (Wallach, 1984). Induction of IL-8 gene expression was determined by Northern analysis. RNA was isolated with TRI kit (Molecular Research Center, Inc.), denatured in formaldehyde/formamide buffer, run through agarose/formaldehyde gel electrophoresis, and then blotted to "GeneScreen Plus" membrane in 10× SSPE buffer On (Du Pont). The above-mentioned processes are all carried out according to conventional techniques. The filters were then hybridized with the IL-8 cDNA probe (Matsushima, et al. 1988, nucleotides No. 1-392). Radioactive labeling (Boehringer Mannheim biochemicala, Mannheim, Germany) was carried out through a random primer box, and strictly washed according to the requirements of the manufacturer. Autoradiography 1-2 days.

(v) Determination of TNF receptor expression

TNF was labeled with ¹²⁵ I according to the chloramine-T method, and the expression level of TNF receptor in a sample containing 1×10 ⁶ cells could be determined by calculating the binding amount with the cells. This method was the same as that described above (Holtmann and Wallach , 1987), the expression of TNF receptors can also be determined by ELISA. That is, according to the method for measuring soluble TNF receptors (Aderka et al., 1991), but the difference is that RIPA buffer (10mM Tris-HCL, pH7.5, 150mM NaCl, 1% NP-40, 1% deoxycholate , 0.1% SDS and 1 mM EDTA) to decompose the cells (70 μl/106 cells) and dilute the test samples. Soluble P55-R purified from urine was used as a standard control.

(b) Detection of segments involved in self-association in P55-IC by mutational analysis of the P55-R intracellular region (P55-IC)

As mentioned above, p55-IC can self-associate and trigger cytotoxic effects, and some fragments of p55-IC can bind full-length p55-IC. In particular, one of the fragments of P55-IC (referred to as protein fragment 55.1 in the above example 1) can combine with the full-length cDNA, and it is found by sequencing it that it contains amino acids between 328-426 of P55-TNF-R residue, and this fragment is also present in P55-IC. Further discoveries (see below). The aforementioned fragment, protein fragment 55.1, is capable of self-association and triggers cytotoxic effects on cells. Therefore, this P55-IC fragment is called "death domain" (or "death region"), and it is located in the segment between 328-426 of human P55-R, which is probably composed of amino acid residue composition.

The fact that the "death domain" of p55-IC has the ability to self-associate was discovered by chance. When using the two-hybrid technique (see Example 1 above) to screen the Hela cell cDNA library for proteins that bind to the intracellular region of the receptor, we found some cDNAs whose encoded products can specifically bind to the intracellular region-GAL4DBD fusion Some of the clones (such as 55.1 and 55.3) encode some fragments of the intracellular region of P55-R (P55-IC, marked with an asterisk in Table 3).

Application of the two-hybrid assay to determine the degree of specificity of P55-IC self-association and to define the relevant segments more precisely led to the following findings (Table 3): (a) The self-association of P55-IC was limited to the "death domain The segment in ", its N-terminus is located in the segment between amino acids 328-344, and its C-terminus is close to the 404th amino acid, that is, the upstream of the reported C-terminus (414th amino acid) of this region. (b) Deletion of the proximal membrane portion upstream of the "death domain" enhanced self-association, suggesting an inhibitory effect of this segment on association. (c) Murine P55-IC can self-associate and also associate with the "death domain" of human P55-R. (d) Three other members of the TNF/NGF receptor family: Fas/Ap01 (FAS-R), CD40 (Field and Song, 1989) and P75 TNF receptors (Smith et al. 1990), self-associate to their intracellular domains Combined studies have shown that: Fas-IC, which can provide a signal for cell death through the sequence motif related to the P55-R "death domain", also has self-association ability, and has a certain degree of interaction with P55-IC association. CD40-IC, however, provides a growth-promoting signal (even though it also includes similar sequences to the "death domain"). In addition, P75-IC has no structural similarity with P55-IC, it cannot self-associate, nor binds to P55-IC or Fas-IC.

table 3

Intracellular domain self-association of P55-R and Fas/AP01 in transformed yeast:

 Determined by a double-hybrid and -galactosidase expression assay

Table 3 on the previous page shows the quantitative test results of the interaction of the Gal4 hybrid constructs. The Gal4 hybrid constructs comprise the following proteins: the intracellular region of human P55-R and its various deletion mutants (residue numbering according to (loetscher et al., 1990 )) (SEQ ID NO: 25); mouse P55-R intracellular region (region between the 334-454th amino acid, numbering according to (Goodwin et al. 1991) (SEQ ID NO: 26)); mouse Fas/Ap01 (Fas -IC, 166-306, numbered according to (Watanabe-Fukanaga et al., 1992)); human CD40 (CD40-IC, 216-277, numbered according to (stamenkovic et al., 1989)); human P75 TNF receptor (P75-IC, 287 -461, numbered according to (smith et al., 1990)). SNF, and SNF4 were used as positive controls for association (Field and Song, 1989), and lamin was used as a negative control (Bartel et al., 1993). Proteins encoded by the Gal4DBD construct (pGPT9) are aligned vertically and proteins encoded by the Gal4AD construct (pGAD-GH) are aligned horizontally. The two deletion mutants marked with an asterisk were cloned in the two-hybrid screening of the HeLa cell cDNA library (Clontech Palo Alto, Ca, USA) by using P55-IC cloned in pGBT9 as "bait" . In the above screening, 4 out of 4×10 ⁶ detected cDNA clones were positive, and it was found that three of them corresponded to certain fragments of human P55-RcDNA (the two clones were identical, encoding between amino acids 328-426 and another region encoding the region between amino acids 277-426). The fourth clone encoded an unknown protein. Expression data for β-galactosidase are the mean of tests of two independent transformants and are listed as the number of β-galactosidase products. (The activity unit is defined as: OD ₄₂₀ × 10 ³ of the enzyme medium divided by the product of OD ₆₀₀ and the reaction time. (OD is the optical density of the yeast culture), and the time is in minutes). The analytical precision is 0.05 units. In all tests, the difference between the same samples was less than 25% of the mean (not tested).

The in vitro test of the interaction between P55-IC glutathione S-transferase (GST) bacterial fusion protein and P55-IC maltose binding protein (MBP) fusion protein confirmed that: P55-R can self-associate, and during the association process Not related to zymogen (see above). Increases in salt concentration did not affect association, nor did EDTA.

In order to assess the significance of the self-association of the "death domain", we investigated the way inducible expression of p55-R or its fragments affects the sensitivity of cells to TNF cytotoxins. The results of the analysis are published in Figure 7, which is expressed by In HeLa cells transfected with P55-R, its intracellular region (P55-IC) or fragments thereof (including the "death domain"), the cytocidal effect has ligand-independent triggering.

In Fig. 7, the following contents are represented in a diagrammatic form: DNA molecules encoding different kinds of TNF receptors, these receptors are present in the vector for transfecting Hela cells (Fig. 7 left most); Utilize the expression vector regulated by tetracycline, Expression of Hela cells transiently expressing various full-length P55-R (P55-R), P55-IC or fragments thereof, or luciferase (luc) as a control (each protein is depicted on the far left of Figure 7) Capacity (left and center tiles) and Viability (right tiles). Empty panels (left, middle, right) represent transfected cells in the presence of expression-inhibiting tetracycline (1 μg/ml); full panels represent transfected cells in the absence of tetracycline. TNF receptor expression was determined 24 hours after transfection by ELISA using antibodies against the extracellular domain of the receptor (illustrated on the left in Figure 7) and also by measuring the binding of radiolabeled TNF to cells ( See middle) The cytocidal effect of transfected proteins was determined 48 hours after transfection. Data presented are from one of three experiments with essentially identical results in which each construct was tested in duplicate. "ND" means "not detected".

It can be seen clearly from Fig. 7: utilize the carrier that can strictly control the expression of transfection cDNA, (the expression of this cDNA is regulated by the reverse transcription protein factor (Gossen and Bujard, 1992) regulated by tetracycline), by encoding full-length Transient expression of the transfected cDNA of the body, a small increase in the expression of P55-R in HeLa cells will lead to extensive cell death. When only P55-IC was expressed, stronger cytotoxicity could be observed. Significant cytotoxicity was also observed in Hela cells when only a fragment of P55-IC containing the essential "death domain" (the region between amino acids 328-426) was expressed. On the other hand, if the fragments of p55-IC lacked or contained only part of the "death domain", their expression had no effect on cell viability (or expressed luciferase gene, used as a control). The cytotoxicity of P55-IC can be further confirmed by using cells stably transformed with P55-IC cDNA; these transformed cells can continue to grow when P55-IC is not induced, but once P55-IC is expressed, they die ( see above).

(c) Other effects of the intracellular region of P55-TNF-R

To investigate whether other activities of TNF could be triggered by self-association of the intracellular domain containing the "death domain"; we investigated the relationship between increased expression of the full-length receptor (P55-R) and the ) expression on the transcription of germicidin 8 (IL-8), which is now known to be activated by TNF (Matsushima et al., 1988). The results are presented in Figure 8, which depicts the ligand-independent induction of IL-8 gene expression in HeLa cells using a tetracycline-regulated construct. Transfected with P55-R or P55-IC (see also "Methods and Materials" and Example 1 above). In column A of Figure 8, the results of Northern blot analysis (see "Methods and Materials" above) are reproduced. /ml, 4 hours) treated (TNF) or not treated (control), or from HTta-1 cells 24 hours after transfection, that is, treated with P55-IC ('P55-IC') P55-R ('P55- R') or luciferase ('luc') transfected cells (in the presence or absence of tetracycline). The results of Northern blot analysis in column B of Figure 8 represent the methylene blue staining of 18SrRNA in each sample in column A.

Thus, it can be seen from Figure 8 that transfection of Hela cells with a tetracycline-regulated construct encoding the cDNA of P55-R induces the transcription of IL-8. While cells were transfected with cDNA encoding P55-IC, a stronger induction could be observed. In both cases, induction occurred only after tetracycline was removed from the cell growth medium, suggesting that induction occurred as a result of expression of transfected p55-R or p55-IC. As a control, transfection of Hela cells with luciferase cDNA had no effect on the transcription of IL-8.

Therefore, from the above results (Fig. 8), only increasing the expression of P55-R, or only increasing the expression of its intracellular domain (P55-IC), triggers cytotoxicity or Among other effects, these include enhancement of intracellular IL-8 gene expression. Triggering of these effects is likely due to self-association of the intracellular domain of P55-R (P55-IC). As mentioned above, it appears that once p55-IC self-associates, its "death domain" will signal the induction of intracellular processes that lead to the triggering of intracellular cytotoxicity, whereas Some other effects, such as a signal to induce IL-8 gene expression, are likely due to the action of other regions of P55-IC after self-association, so it is likely that different regions of P55-IC are responsible for different intracellular TNF-induced effects ( Such as cytotoxicity, induction of IL-8), these effects are the result of intracellular signal initiation after p55-IC self-association.

The fact that p55-IC can induce the triggering of other intracellular effects, such as IL-8, in a ligand-independent (TNF)-independent manner suggests that p55-IC, or specific parts thereof, can be used as specific tools in Such effects are brought about in cells or tissues that need to be treated, and these tissues or cells need not be treated with TNF. In many pathological conditions such as tumors, treatment with TNF, especially at high doses, may lead to undesirable side effects due to several intracellular effects induced systemically by binding of TNF to receptors. The present invention's discovery that P55-IC can also mimic specific other TNF-inducing effects (besides cytotoxicity), such as the induction of IL-8 expression, will open up a new approach that allows p55-IC or its specific The fragments are introduced into specific cells or tissues, and can be used as signals to induce specific ideal intracellular effects, such as the induction of IL-8, thus overcoming the common systemic side effects in TNF treatment.

(d) Ligand-independent triggering of the cytocidal effect of P55TNF-R and FAS-R (Fas/Ap01) intracellular region and its "death domain" in Hela cells.

The cytotoxicity of the intracellular regions of P55TNF-R and FAS-R (P55IC and FAS-IC) is now further elucidated: P55-IC, its "death domain" (P55DD) and FAS-IC in Hela cells Both have ligand-independent triggering of cytocidal effects. In this study, HeLa cells were transfected with expression vectors. These expression vectors contain different constructs: constructs containing full-length P55-TNF-R, including fragments of P55IC and P55DD; constructs containing FAS-IC. In one set of experiments, Hela cells were transfected with a P55TNF-R-containing construct and a FAS-IC-containing construct (see above for details of the constructs, preparation, etc.). The results of this study are presented in Figure 9 (A and B), where the constructs used to transfect Hela cells are listed schematically in the left column. Results for FAS or TNF receptor expression are presented graphically in the middle two columns (second and third from left) and for transfected cell viability in the right column. In Figure 9A, the results of transfected Hela cells transiently expressing full-length P55-R, P55-IC or fragments thereof, or luciferase (luc) as a control are shown. It is a tetracycline-regulated expression vector. In Fig. 9B, the results of transfected Hela cells expressing FAS-IC alone or P55-R and FAS-IC at the same time are shown, and the expression vector regulated by tetracycline was also used. In the graphical results of Figures 9A and B, open blocks represent transfected cells in the presence of expression-suppressing tetracycline (1 μg/ml) and closed blocks represent transfected cells in the absence of expression-suppressing tetracycline. TNF receptor expression was measured 20 hours after transfection, either by ELISA using antibodies against the extracellular domain of the receptor (see left column) or by measuring radiolabeled TNF receptor expression was determined by the amount of TNF bound to the cells (middle column). The cytocidal effect of transfected proteins was determined 48 hours after transfection. Published data are from one of three experiments with essentially identical results in which each construct was tested in duplicate. References to "ND" in Figures 9A and B indicate that no detection was performed. It is evident from the results in Figures 9A and B that the expression of P55IC alone resulted in stronger cytotoxicity. Significant cytotoxicity was also produced when only the "death domain" was expressed, in contrast, the expression of some fragments of p55IC lacking the "death domain" or containing only the "death domain" fragment had no effect on cell survival. The expression of FAS-IC did not lead to meaningful cytotoxicity, but rather significantly enhanced the cytotoxicity of its expressed P55-R.

Example 3

Additional proteins capable of binding to the intracellular domain of P55TNF-R or FAS-R

Using the same methods and techniques as in the above example, three other proteins have been isolated and identified as binding to p55IC or FAS-IC.

In Figures 10-12, part of the basic nucleotide sequences of three cDNA clones, designated F2, F9 and DD11, are published in schematic form.

Using the mouse FAS-IC as "bait", the mouse embryo library was screened, and F2 clone and F9 clone were isolated from it. In Fig. 10, the partial nucleotide sequence of the F2cDNA that has measured the sequence is shown in graphic form, and the nucleotide sequence that contains 1724 bases from the F9cDNA that has measured the sequence is shown in graphic form in Fig. 11 part. The binding abilities of the proteins encoded by the F2 and F9 clones were analyzed separately, showing that:

(a) F2 strongly interacts with human P55IC and P55DD and murine FAS-IC, whereas it interacts with the related protein, human FAS-IC, and with the unrelated (control) proteins SNF ₁ and lamin. very weak.

(b) F9 strongly interacts with human P55-IC and P55DD and murine FAS-IC, but weakly with human FAS-IC (associated protein) and unrelated proteins SNF1 and lamin.

(c) Neither F2 nor F9 had any interaction with human P75IC, pGBT9 (empty vector) or human CD-40.

In addition, searches from "Gene Bank" and "Protein Bank" indicated that F2 and F9 represented two novel proteins.

Thus, F2 and F9 represent two novel proteins with binding specificity for FAS-IC and P55IC.

Human P55DD was used as "bait" to screen the human Hela library, and DD11 clone was isolated therefrom. Figure 12 shows a part of the 425-base nucleotide sequence from DD11 cDNA of known sequence in a schematic form.

The DD11 clone contains approximately 800 nucleotides. Using this clone as a probe, the full length of its transcript was found to be about 1200 bases. Analysis of the binding ability of the protein encoded by the DD11 clone showed that DD11 had a strong interaction with P55DD (amino acid 326-414) (see Figure 9), but had no interaction with deletion mutants of this region, such as amino acid 326-404 effect. DD11 can interact with human and mouse FAS-IC, and also interacts with nuclear lamin to some extent. DD11 did not interact with SNF at all, nor with pGBT9 (empty bait vector). DD11 was not found in either the "gene pool" or the "protein pool". Therefore, DD11 is a protein that can specifically bind to P55IC (P55DD) and FAS-IC.

Example 4

Construction of Soluble Dimeric TNF Receptor

Based on the findings in Example 2 above, the intracellular region of P55-R (P55-IC) and fragments thereof ("death domain"), the intracellular region of Fas/AP01 and fragments thereof (also referred to as the "death domain") ) have self-association ability. It is therefore possible to construct new TNF receptors which will have the ability to self-associate (aggregate) and be soluble. Such a TNF receptor should be a fusion protein, that is, a fusion product of the entire extracellular region containing the necessary P55-R and the entire intracellular region containing the necessary P55-R or Fas/AP01 or its "death function domain". Thus, such fusion constructs will lack the transmembrane region of P55-R, so they are soluble. Furthermore, due to the self-association ability of the intracellular region or its "death domain", these fusion constructs will have the ability to oligomerize and form at least dimers (and possibly higher order multimers) of P55-R. body). As a result, these dimeric TNF receptors (P55-R) will be able to bind at least two TNF monomers of the naturally occurring TNF homotrimer and provide a receptor that is better able to bind to its ligand (homotrimeric TNF). Binding of soluble TNF receptors.

Therefore, it is necessary to construct at least four types of P55TNF receptor fusion proteins, each of which has oligomerization ability and is soluble.

(i) fusion protein composed of P55-R extracellular region (EC55) and P55-R intracellular region (p55-IC);

(ii) a fusion protein composed of EC55 and the "death domain" (DD55) of p55-IC;

(iii) a fusion protein composed of EC55 and Fas/Ap01 intracellular region (ICAS); and

(iv) A fusion protein composed of EC55 and the "death functional domain" (DDFAS) of ICFAS.

In each of the above fusion proteins, the binding ability of TNF monomer is produced by the EC55 part, while the oligomerization ability (or at least dimerization ability) is produced by the "tail region", which is the p55IC part, or the DD55 part , or ICFAS section, or DDAS section.

In order to construct the above-mentioned fusion protein, it is necessary to apply the conventional technique of recombinant DNA, which is now well established (see: Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Experimental Press, Cold Spring Harbor: New York) Briefly, any suitable bacterial, phage or animal viral expression vector (cloning vector or plasmid designed for selective insertion of DNA expression) can be used in one or more stages to insert the DNA encoding EC55 and one of the "tail regions" . The "tail region" is p55-IC, or DD55, or ICFAS, or DDFAS. Insert DNA encoding each fusion protein will be placed under various expression control sequences of the cloning vector or plasmid, such as promoter, ribozyme binding site, transcription factor binding site, etc. The selection of the expression control sequence depends on the type of expression vector selected and also on the type of host cell (eukaryotic or prokaryotic), so that the fusion protein of the present invention can be better expressed. Ideal host cells (and vectors) are eukaryotic cell types, especially mammalian cells.

DNA molecules encoding each of the above fusion proteins were prepared and inserted into expression vectors according to the following procedures.

(a) at first, construct the oligonucleotide used in the PCR according to conventional method, these oligonucleotides represent as follows:

1) ACC ATG GGC CTC TCC ACC GTG (EC55, positive sense chain) (SEQ ID NO: 1)

2) ACGC GTC GAC TGT GGT GCC TGA GTC CTC (EC55, antisense chain) (SEQ ID NO: 2)

3) ACGC GTC GAC CGC TAC CAA CGG TGG AAG (TC55, positive sense chain) (SEQ ID NO: 3)

4) TCA TCT GAG AAG ACT GGG (IC55, antisense chain) (SEQ ID NO: 4)

5) ACGC GTC GAC AAG AGA AAG GAA GTA CAG (IC FAS, positive meaning chain) (SEQ IDNO: 5)

6) CTA GAC CAA GCT TTG GAT (IC FAS, antisense chain) (SEQ ID NO: 6)

7) ACGC GTC GAC CCC GCG ACG CTG TAC GCG (DD55, positive sense chain) (SEQ ID NO: 7)

8) ACGC GTC GAC GAT GTT GAC TTG AGT AAA (DD FAS, positive sense chain) (SEQ ID NO: 8)

b) Our laboratory has plasmids containing full-length p55-R and Fas/Ap01 receptor-encoding clones (see EP568925 and examples 1-3 above), which are used for the following amplifications to produce fusion proteins encoding each DNA fragments, and connect these DNA fragments into the above-mentioned selected expression vectors.

(i) In order to synthesize a DNA fragment encoding EC55 as one of the four fusion protein components, use the above-mentioned oligonucleotides 1 and 2 as primers to perform PCR amplification on a plasmid containing human P55 cDNA (about 640 bases in size) base pair fragments).

(ii) In order to obtain the fusion product EC55-IC55, use the above oligonucleotides 3 and 4 as primers to perform PCR amplification on the plasmid containing human p55cDNA to obtain a DNA fragment encoding IC55 (about 677 base pairs in size) , and then the fragment was mixed with saLI-digested EC55, and then inserted into a mammalian cell expression vector under the control of a suitable promoter by blunt-end ligation. The insertion direction of IC55-EC55 in the vector was identified by restriction enzyme digestion and sequencing techniques.

(iii) In order to obtain the fusion product EC55-ICFAS, use oligonucleotides 5 and 6 as primers to carry out PCR amplification on the plasmid containing the FAScDNA clone to obtain a DNA fragment (about 448 base pairs in size) encoding ICFAS, and then The fragment was digested with SalI and mixed with the EC55 digested with SalI, and then inserted into a mammalian cell expression vector under the control of a suitable promoter by blunt-end ligation. The insertion direction of EC55-ICFAS in the vector was identified by restriction enzyme digestion and sequencing techniques.

(iv) In order to obtain the fusion product EC55-DD55, use oligonucleotides 7 and 4 as primers to carry out PCR amplification on human P55cDNA to obtain a DNA fragment with a DD55 coding sequence, which is about 314 base pairs in size. Digested with SalI and mixed with EC55 digested with SalI, and then ligated into a mammalian cell expression vector by blunt-end ligation. The insertion direction of EC55-DD55 in the vector was identified by restriction enzyme cutting technology and sequencing technology.

(V) In order to obtain the fusion product EC55-DDFAS, use oligonucleotides 6 and 8 as primers to perform PCR amplification on the FAScDNA clone to obtain a DNA fragment containing the DDFAS coding sequence, with a size of about 332 base pairs. Digested with SalI and mixed with EC55 digested with SalI, and then ligated into a mammalian cell expression vector by blunt-end ligation. The insertion direction of EC55-DDFAS in the vector was identified by restriction enzyme digestion and sequencing techniques.

Once the above expression vectors are constructed, they can be introduced into suitable mammalian cells (such as Chinese hamster ovary cells (CHO) or monkey kidney cells (COS)) by conventional techniques for expression. The expressed fusion protein will be purified by conventional techniques (see EP308378, EP398327 and EP568925). Purified proteins were analyzed for their ability to oligomerize (and their extent, i.e., whether they form dimers or higher multimers), as well as for their ability to bind TNF (and their binding affinity) analyzed.

Example 5

Construction of Soluble Dimeric Fas/Ap01 Receptor

In the same manner as in Example 4 above, the following four types of Fas/Ap01 fusion products can be synthesized, each of which has oligomerization ability and is soluble:

(i) fusion product formed between the extracellular region of Fas/Ap01 (ECFAS) and the intracellular region of P55-IC;

(ii) The fusion product formed between ECFAS and the "death domain" (DD55) of P55-IC;

(iii) a fusion product formed between ECFAS and the Fas/Ap01 intracellular region (ICFAS);

(iv) A fusion product formed between ECFAS and the "death functional domain" (DDFAS) of ICFAS.

In each of the above fusion proteins, the FAS ligand-binding ability is generated by the ECFAS part; and the oligomerization ability (at least dimerization ability) is generated by the "tail region", which is P55-IC, or DD55, or ICFAS , or any of the DDFAS sections.

The construction of the DNA fragment encoding the above fusion protein and its expression vector is the same as Example 4, but here a different oligonucleotide sequence (unpublished) is used to prepare the ECFAS fragment and connect to any of the above "tail regions". Next, the expression vector is introduced into a suitable host cell, and the expressed fusion proteins are purified and then analyzed for their ability to oligomerize (and their extent, ie, whether they form dimers or higher-order multimers). Their ability to bind to the FAS ligand (and their binding affinity) was also analyzed.

Example 6

Construction of a soluble oligomeric "hybrid" TNF/FAS receptor

In order to prepare oligomeric receptors with "mixed" affinity, i.e., receptors with affinity for both TNF and FAS-R ligands, the fusion products mentioned above (Examples 4 and 5) will be applied to the following program:

i) Synthesizing a fusion product as described in Example 4, which comprises the extracellular region of TNF-R (P75TNF-R or P55TNF-R) fused to any of these four proteins, P55IC, FAS-IC, P55DD, and FASDD A sort of.

ii) Synthesizing a fusion product as described in Example 5, which comprises the fusion of the extracellular region of TNF-R to any one of the four proteins P55IC, P55DD, FAS-IC, and FAS-DD.

iii) Mix any fusion product in i) with any fusion product in ii) to obtain a new dimer receptor (or higher multimer), which has both TNF-R and FAS- The extracellular region of R. These two extracellular regions are connected by -IC region or -DD segment.

In the above procedure, the fusion product in i) and the fusion product in ii) can be synthesized separately, that is, first purified from the transformed cells producing them, and then mixed in vitro to obtain mixed affinity receptors. In addition, host cells can be co-transfected with a vector containing the coding sequence of the fusion product. In this case, mixed affinity receptors can be directly obtained from the transfected host cells. The actual oligomerization of the fusion product to become an oligomerization receptor can occur in the cell, or during the purification process of the fusion product, or after purification, so that the fusion product expressed in the cell can be obtained. In order to accurately select mixed affinity receptors, various conventional techniques can be applied, for example, the application of affinity chromatography, in which antibodies against the extracellular domains of TNF-R and FAS-R are used in consecutive Receptors with both extracellular domains are selected in the chromatographic step.

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Sequence Listing

(1) General information:

(i) Applicant:

(A) Name: Yeda Research and Development Co., Ltd.

(B) Street: P.O. Box 95

(C) City: Rehovot

(E) Country: Israel

(F) Zip Code (ZIP): 76100

(G) Tel: 011-972-847-0617

(H) Telex: 011-972-847-0733

(A) Name: Henry WEINWURZEL

(B) Street: Herzl Street 43

(C) City: Raanana

(E) Country: Israel

(F) Zip code (ZIP): 43353

(A) Name: David WALLACH

(B) Street: 24 Borochov Street

(C) City: Rehovot

(E) Country: Israel

(F) Zip Code (ZIP): 76406

(A) Name: Mark BOLDIN

(B) Street: Zelenograd 927-53

(C) City: Moscow

(E) Country: Russia

(F) Zip code (ZIP): 103575

(A) Name: Igor METT

(B) Street: 60 Levin Epstein Street

(C) City: Rehovot

(E) Country: Israel

(F) Zip Code (ZIP): 76462

(A) Name: Eugene VARFOLOMEEV

(B) Street: P.O.Box 26

(C) City: Rehovot

(E) Country: Israel

(F) Zip Code (ZIP): 76100

(ii) Title of invention: TNF/NGF superfamily receptors and modulators of soluble oligomeric TNF/NGF superfamily receptors

(iii) Number of sequences: 26

(iv) Mailing address:

(A) Recipient: BROWDY AND NEIMARK

(B) Street: 419 Seventh Street, N.W., Suite 300

(C) City: Washington

(D) State: D.C.

(E) Country: USA

(F)ZIP: 20004

(v) In computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM PC compatible

(C) Operating system: PC-DOS/MS-DOS

(D) Software: PatentIn Release#1.0, Version#1.30(EPO)

(v) Current application information:

  Application No.: PCT/US95/05854

(vi) Prior application materials:

(A) Application number: IL 109,632

(B) Filing date: 11-MAY-1994

(vi) Prior application materials:

(A) Application number: IL 111,125

(B) Filing date: 02-OCT-1994

(viii) Lawyer/Representative Information:

(A) Name: BROWDY, Roger L.

(B) Registration number: 25,618

(C) Reference/File number: WALLACH＝15 PCT

(ix) Telecommunication information:

(A) Tel: 202-628-5197

(B)Tel: 202-737-3528

(C) Telegram: 248633

(2) SEQ ID NO: 1 information:

(i) Sequence features:

(A) Length: 21bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 1:

ACCATGGGCC TCTCCACCGT G 21

(2) Information SEQ ID NO: 2:

(i) Sequence features:

(A) Length: 28bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

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

ACGCGTCGAC TGTGGTGCCT GAGTCCTC 28

(2) Information SEQ ID NO: 3:

(i) Sequence features:

(A) Length: 28bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 3:

ACGCGTCGAC CGCTACCAAC GGTGGAAG 28

(2) Information SEQ ID NO: 4:

(i) Sequence features:

(A) Length: 18bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 4:

TCATCTGAGA AGACTGGG 18

(2) Information SEQ ID NO: 5:

(i) Sequence features:

(A) Length: 28bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 5:

ACGCGTCGAC AAGAGAAAGG AAGTACAG 28

(2) Information SEQ ID NO: 6:

(i) Sequence features:

(A) Length: 18bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

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

CTAGACCAAG CTTTGGAT 18

(2) Information SEQ ID NO: 7:

(i) Sequence features:

(A) Length: 28bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 7:

ACGCGTCGAC CCCGCGACGC TGTACGCC 28

(2) Information SEQ ID NO: 8:

(i) Sequence features:

(A) Length: 28bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 8:

ACGCGTCGAC GATGTTGACT TGAGTAAA 28

(2) Information SEQ ID NO: 9:

(i) Sequence features:

(A) Length: 2866bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 9:

ATTCGGGTGC AGCCCCAGCA GTCTCCAGCG GCGGCCCCCG GCGGCACGGA CGAGAAGCCG 60

AGCGGCAAGG AGCGGCGGGA TGCCGGGGAC AAGGACAAAG AACAGGAGCT GTCTGAAGAG 120

GATAAACAGC TTCAAGATGA ACTGGAGATG CTCGTGGAAC GACTAGGGGA GAAGGATACA 180

TCCCTGTATC GACCAGCGCT GGAGGAATTG CGAAGGCAGA TTCGTTCTTC TACAACTTCC 240

ATGACTTCAG TGCCCAAGCC TCTCAAATTT CTGCGTCCAC ACTATGGCAA ACTGAAGGAA 300

ATCTATGAGA ACATGGCCCC TGGGGAGAAT AAGCGTTTTG CTGCTGACAT CATCTCCGTT 360

TTGGCCATGA CCATGAGTGG GGAGCGTGAG TGCCTCAAGT ATCGGCTAGT GGGCTCCCAG 420

GAGGAATTGG CATCATGGGG TCATGAGTAT GTCAGGCATC TGGCAGGAGA AGTGGCTAAG 480

GAGTGGCAGG AGCTGGATGA CGCAGAGAAG GTCCAGCGGG AGCCTCTGCT CACTCTGGTG 540

AAGGAAATCG TCCCCTATAA CATGGCCCAC AATGCAGAGC ATGAGGCTTG CGACCTGCTT 600

ATGGAAATTG AGCAGGTGGA CATGCTGGAG AAGGACATTG ATGAAAATGC ATATGCAAAG 660

GTCTGCCTTT ATCTCACCAG TTGTGTGAAT TACGTGCCTG AGCCTGAGAA CTCAGCCCTA 720

CTGCGTTGTG CCCTGGGTGT GTTCCGAAAG TTTACCCGCT TCCCTGAAGC TCTGAGATTG 780

GCATTGATGC TCAATGACAT GGAGTTGGTA GAAGACATCT TCACCTCCTG CAAGGATGTG 840

GTAGTACAGA AACAGATGGC ATTCATGCTA GGCCGGCATG GGGTGTTCCT GGAGCTGAGT 900

GAAGATGTCG AGGAGTATGA GGACCTGACA GAGATCATGT CCAATGTACA GCTCAACAGC 960

AACTTCTTGG CCTTAGCTCG GGAGCTGGAC ATCATGGAGC CCAAGGTGCC TGATGACATC 1020

TACAAAACCC ACCTAGAGAA CAACAGGTTT GGGGGCAGTG GCTCTCAGGT GGACTCTGCC 1080

CGCATGAACC TGGCCTCCTC TTTTGTGAAT GGCTTYGTGA ATGCAGCTTT TGGCCAAGAC 1140

AAGCTGCTAA CAGATGATGG CAACAAATGG CTTTACAAGA ACAAGGACCA CGGAATGTTG 1200

AGTGCAGCTG CATCTCTTGG GATGATTCTG CTGTGGGATG TGGATGGTGG CCTCACCCAG 1260

ATTGACAAGT ACCTGTACTC CTCTGAGGAC TACATTAAGT CAGGAGCTCT TCTTGCCTGT 1320

GGCATAGTGA ACTCTGGGGT CCGGAATGAG TGTGACCCTG CTCTGGCACT GCTCTCAGAC 1380

TATGTTCTCC ACAACAGCAA CACCATGAGA CTTGGTTCCA TCTTTGGGCT AGGCTTGGCT 1440

TATGCTGGCT CAAATCGTGA AGATGTCCTA ACACTGCTGC TGCCTGTGAT GGGAGATTCA 1500

AAGTCCAGCA TGGAGGTGGC AGGTGTCACA GCTTTAGCCT GTGGAATGAT AGCAGTAGGG 1560

TCCTGCAATG GAGATGTAAC TTCCACTATC CTTCAGACCA TCATGGAGAA GTCAGAGACT 1620

GAGCTCAAGG ATACTTATGC TCGTTGGCTT CCTCTTGGAC TGGGTCTCAA CCACCTGGGG 1680

AAGGGTGAGG CCATCGAGGC AATCCTGGCT GCACTGGAGG TTGTGTCAGA GCCATTCCGC 1740

AGTTTTGCCA ACACACTGGT GGATGTGTGT GCATATGCAG GCTCTGGGAA TGTGCTGAAG 1800

GTGCAGCAGC TGCTCCACAT TTGTAGCGAA CACTTTGACT CCAAAGAGAA GGAGGAAGAC 1860

AAAGACAAGA AGGAAAAGAA AGACAAGGAC AAGAAGGAAG CCCCTGCTGA CATGGGAGCA 1920

CATCAGGGAG TGGCTGTTCT GGGGATTGCC CTTATTGCTA TGGGGGAGGA GATTGGTGCA 1980

GAGATGGCAT TACGAACCTT TGGCCACTTG CTGAGATATG GGGAGCCTAC ACTCCGGAGG 2040

GCTGTACCTT TAGCACTGGC CCTCATCTCT GTTTCAAATC CACGACTCAA CATCCTGGAT 2100

ACCCTAAGCA AATTCTCTCA TGATGCTGAT CCAGAAGTTT CCTATAACTC CATTTTTGCC 2160

ATGGGCATGG TGGGCAGTGG TACCAATAAT GCCCGTCTGG CTGCAATGCT GCGCCAGTTA 2220

GCTCAATATC ATGCCAAGGA CCCAAACAAC CTCTTCATGG TGCGCTTGGC ACAGGGCCTG 2280

ACACATTTAG GGAAGGGCAC CCTTACCCTC TGCCCCTACC ACAGCGACCG GCAGCTTATG 2340

AGCCAGGTGG CCGTGGCTGG ACTGCTCACT GTGCTTGTCT CTTTCCTGGA TGTTCGAAAC 2400

ATTATTCTAG GCAAATCACA CTATGTATTG TATGGGCTGG TGGCTGCCAT GCAGCCCCGA 2460

ATGCTGGTTA CGTTTGATGA GGAGCTGCGG CCATTGCCAG TGTCTGTCCG TGTGGGCCAG 2520

GCAGTGGATG TGGTGGGCCA GGCTGGCAAG CCGAAGACTA TCACAGGGTT CCAGACGCAT 2580

ACAACCCCAG TGTTGTTGGC CCACGGGGAA CGGGCAGAAT TGGCCACTGA GGAGTTCCTT 2640

CCTGTTACCC CCATTCTGGA AGGTTTTGTT ATCTTCGGAA GAACCCCAAT TATGATCTCT 2700

AAGTGACCAC CAGGGGCTCT GAACTGCAGC TGATGTTATC AGCAGGACAT GCATCCTGCT 2760

GCCAAGGGTG GACACGGCTG CAGACTTCTG GGGGAATTGT CGCCTCCTGC TCTTTTGTTA 2820

CTGAGTGAGA TAAGGTTGTT CAATAAAGAC TTTTATCCCC AAGGTC 2866

(2) Information SEQ ID NO: 10:

(i) Sequence features:

(A) Length: 676bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 10:

GAATTCGGCA CGAGCGGCAC GAGGACAGAG TGAGACTCTG TCTCTTAAAA TAATAATAAA 60

AATAAAAATA AAATGTGGGG CCGGGCAAGG TGGCTCATGC CTGTAATCCC AGCACCTTGG 120

GAGGCTGAGG CAGGAGGATT GCCTAAAGCCC AGGAGTTTGA CATCAGCCTG GGCAACATGG 180

TGAAACCCCA TCTCTACAAA AAATGCAAAA ATTAGCCAGG TGTGGTGGGT GTGCTCCTAT 240

AGTCTCAGCT ACTCAGGAGG CTGAGGTAGA GGGGATCACC TGAGCCCAGG AAGTTTGGAG 300

GCTATAGTGA GCTGAAGACC CGCACCATTG CACGCCAGCC TGGAGCAAGA GACNCTGTCT 360

CCACATAAAT AAATAAATAA ATAAAAGTGG GGAACTTCTG TGTTAAGTCA GAAGGCACCA 420

CACAATTTGN ATAGCCANCA ACCATATTCA ATACCCAATC TCTTTATTGC AATATAAGTA 480

TTTGTAAACC CCTACACAAA TATTCCCAAG AATAAGTTGG AATATAAATT ACTATATCAA 540

TCANCCAATA AAAATAAACA CATACAGTAT TTATTTCCTG TTGCTCCATA TAAAGCTTTG 600

CTATTTCAAT ATAAAGCTTA CCTAGTATGG TCATTTGAGC CTGAGCAGAG AATATGCCCA 660

AGCTCGTGCC GAATTC 676

(2) Information SEQ ID NO: 11:

(i) Sequence features:

(A) Length: 1153bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 11:

GGCGNTCTGA CTCTCTACTG AACCAAGACT GAATCAGAGA GACTCGAGTG CNCTTATTTG 60

ATTAANCCCA AATTATTGAA ACCTNTGATT TTTTCTGGAG GNGGATGATA AAGATGTGAA 120

AGTGTGATGA ACAGTGTGTA TCCCTACTCT TGATCCTGGA ACCAGACAAG CAAGAAGCTT 180

TGATTGAAAG CCTATGTGAA AAGCTGGTCA AATTTCGCGA AGGTGAACGC CCGTCTCTGA 240

GACTGCAGTT GTTAAGCAAC CTTTTCCACG GGATGGATAA GAATACTCCT GTAAGATACA 300

CAGTGTATTG CAGCCTTATT AAAGTGGCAG CATCTTGTGG GGCCATCCAG TACATCCCAA 360

CTGAGCTGGA TCAAGTTAGA AAATGGATTT CTGACTGGAA TTCTCACCACT GAAAAAAAGC 420

ACACCCTTTT AAGACTACTT TATGAGGCAC TTGTGGATTG TAAGAAGAGT GATGCTGCTT 480

CAAAAGTCAT GGTGGAATTG CTCGGAAGTT ACACAGAGGA CAATGCTTCC CAGGCTCGAG 540

TTGATGCCCA CAGGTGTATT GTACGAGCAT TGAAAGATCC AAATGCATTT CTTTGTGACC 600

ACCTTCTTAC TTTAAAACCA GTCAAGTTTG TGGAAGGCGA GCTTATTCAT GATCTTTTAA 660

CCATTTGTGT GAGTGCTAAA TTGGCATCAT ATGTCAAGTT TTATCAGAAT AATAAAGACT 720

TCATTGATTC ACTTGGCCTG TTACATGAAC AGAATATGGC AAAAATGAGA CTACTTACTT 780

TTATGGGAAT GGCAGTAGAA AATAAGGAAA TTTCTTTTGA CACAATGCAG CAAGAACTTC 840

AGATTGGAGC TGATGATGTT GAAGCATTTG TTATTGACGC CGTAAGAACT AAAATGGTCT 900

ACTGCAAAAT TGATCAGACC CAGAGAAAAG TAGTTGTCAG TCATAGCACA CATCGGACAT 960

TTGGAAAACA GCAGTGGCAA CAACTGTATG ACACACTTAA TGCCTGGAAA CAAAATCTGA 1020

ACAAAGTGAA AAACAGCCTT TTGAGTCTTT CTGATACCTG AGTTTTTTATG CTTATAATTT 1080

TTGTTCTTTG AAAAAAAAGC CCTAAATCAT AGTAAAACAT TATAAACTAA AAAAAAAAAA 1140

AAAAAAACTC GAG 1153

(2) Information SEQ ID NO: 12:

(i) Sequence features:

(A) Length: 220bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 12:

GTCCGGTTTA CTTTAACTTA GTTTTGCATA GTTCTAGTGC ACGTGAAATT GAAAAGTTAT 60

TTCCCTTTAG CTGTGTTATT ATAGAGCAGA AATTCTGTTT TTAAAAAATTA GCCTAAGATA 120

TACTTGTTTT TGTAAAGAAA AATATTTAAT GCTTGAACAA AATAAATTGG AGTTGGAGTA 180

GAATGTAGTT TGAGGAAATT TGCAGCTTCC AATGCCTCTG 220

(2) Information SEQ ID NO: 13:

(i) Sequence features:

(A) Length: 220bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 13:

CAGAGGCATT GGAAGCTGCA AATTTCCTCA AACTACATTC TACTCCAACT CCAATTTATT 60

TTGTTCAAGC ATTAAATATT TTTCTTTACA AAAACAAGTA TATCTTAGGC TAATTTTTAA 120

AAACAGAATT TCTGCTCTAT AATAACACAG CTAAAGGGAA ATAACTTTTC AATTTCACGT 180

GCACTAGAAC TATGCAAAAC TAAGTTAAAG TAAACCGGAC 220

(2) Information SEQ ID NO: 14:

(i) Sequence features:

(A) Length: 900AA

(B) type: amino acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 14:

Arg Val Gln Pro Gln Gln Ser Pro Ala Ala Ala Pro Gly Gly Thr Asp

1 5 10 15

Glu Lys Pro Ser Gly Lys Glu Arg Arg Asp Ala Gly Asp Lys Asp Lys

20 25 30

Glu Gln Glu Leu Ser Glu Glu Asp Lys Gln Leu Gln Asp Glu Leu Glu

35 40 45

Met Leu Val Glu Arg Leu Gly Glu Lys Asp Thr Ser Leu Tyr Arg Pro

50 55 60

Ala Leu Glu Glu Leu Arg Arg Gln Ile Arg Ser Ser Thr Thr Ser Met

65 70 75 80

Thr Ser Val Pro Lys Pro Leu Lys Phe Leu Arg Pro His Tyr Gly Lys

85 90 95

Leu Lys Glu Ile Tyr Glu Asn Met Ala Pro Gly Glu Asn Lys Arg Phe

100 105 110

Ala Ala Asp Ile Ile Ser Val Leu Ala Met Thr Met Ser Gly Glu Arg

115 120 125

Glu Cys Leu Lys Tyr Arg Leu Val Gly Ser Gln Glu Glu Leu Ala Ser

130 135 140

Trp Gly His Glu Tyr Val Arg His Leu Ala Gly Glu Val Ala Lys Glu

145 150 155 160

Trp Gln Glu Leu Asp Asp Ala Glu Lys Val Gln Arg Glu Pro Leu Leu

165 170 175

Thr Leu Val Lys Glu Ile Val Pro Tyr Asn Met Ala His Asn Ala Glu

180 185 190

His Glu Ala Cys Asp Leu Leu Met Glu Ile Glu Gln Val Asp Met Leu

195 200 205

Glu Lys Asp Ile Asp Glu Asn Ala Tyr Ala Lys Val Cys Leu Tyr Leu

210 215 220

Thr Ser Cys Val Asn Tyr Val Pro Glu Pro Glu Asn Ser Ala Leu Leu

225 230 235 240

Arg Cys Ala Leu Gly Val Phe Arg Lys Phe Thr Arg Phe Pro Glu Ala

245 250 255

Leu Arg Leu Ala Leu Met Leu Asn Asp Met Glu Leu Val Glu Asp Ile

260 265 270

Phe Thr Ser Cys Lys Asp Val Val Val Gln Lys Gln Met Ala Phe Met

275 280 285

Leu Gly Arg His Gly Val Phe Leu Glu Leu Ser Glu Asp Val Glu Glu

290 295 300

Tyr Glu Asp Leu Thr Glu Ile Met Ser Asn Val Gln Leu Asn Ser Asn

305 310 315 320

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

325 330 335

Asp Asp Ile Tyr Lys Thr His Leu Glu Asn Asn Arg Phe Gly Gly Ser

340 345 350

Gly Ser Gln Val Asp Ser Ala Arg Met Asn Leu Ala Ser Ser Phe Val

355 360 365

Asn Gly Phe Val Asn Ala Ala Phe Gly Gln Asp Lys Leu Leu Thr Asp

370 375 380

Asp Gly Asn Lys Trp Leu Tyr Lys Asn Lys Asp His Gly Met Leu Ser

385 390 395 400

Ala Ala Ala Ser Leu Gly Met Ile Leu Leu Trp Asp Val Asp Gly Gly

405 410 415

Leu Thr Gln Ile Asp Lys Tyr Leu Tyr Ser Ser Glu Asp Tyr Ile Lys

420 425 430

Ser Gly Ala Leu Leu Ala Cys Gly Ile Val Asn Ser Gly Val Arg Asn

435 440 445

Glu Cys Asp Pro Ala Leu Ala Leu Leu Ser Asp Tyr Val Leu His Asn

450 455 460

Ser Asn Thr Met Arg Leu Gly Ser Ile Phe Gly Leu Gly Leu Ala Tyr

465 470 475 480

Ala Gly Ser Asn Arg Glu Asp Val Leu Thr Leu Leu Leu Pro Val Met

485 490 495

Gly Asp Ser Lys Ser Ser Met Glu Val Ala Gly Val Thr Ala Leu Ala

500 505 510

Cys Gly Met Ile Ala Val Gly Ser Cys Asn Gly Asp Val Thr Ser Thr

515 520 525

Ile Leu Gln Thr Ile Met Glu Lys Ser Glu Thr Glu Leu Lys Asp Thr

530 535 540

Tyr Ala Arg Trp Leu Pro Leu Gly Leu Gly Leu Asn His Leu Gly Lys

545 550 555 560

Gly Glu Ala Ile Glu Ala Ile Leu Ala Ala Leu Glu Val Val Ser Glu

565 570 575

Pro Phe Arg Ser Phe Ala Asn Thr Leu Val Asp Val Cys Ala Tyr Ala

580 585 590

Gly Ser Gly Asn Val Leu Lys Val Gln Gln Leu Leu His Ile Cys Ser

595 600 605

Glu His Phe Asp Ser Lys Glu Lys Glu Glu Asp Lys Asp Lys Lys Glu

610 615 620

Lys Lys Asp Lys Asp Lys Lys Glu Ala Pro Ala Asp Met Gly Ala His

625 630 635 640

Gln Gly Val Ala Val Leu Gly Ile Ala Leu Ile Ala Met Gly Glu Glu

645 650 655

Ile Gly Ala Glu Met Ala Leu Arg Thr Phe Gly His Leu Leu Arg Tyr

660 665 670

Gly Glu Pro Thr Leu Arg Arg Ala Val Pro Leu Ala Leu Ala Leu Ile

675 680 685

Ser Val Ser Asn Pro Arg Leu Asn Ile Leu Asp Thr Leu Ser Lys Phe

690 695 700

Ser His Asp Ala Asp Pro Glu Val Ser Tyr Asn Ser Ile Phe Ala Met

705 710 715 720

Gly Met Val Gly Ser Gly Thr Asn Asn Ala Arg Leu Ala Ala Met Leu

725 730 735

Arg Gln Leu Ala Gln Tyr His Ala Lys Asp Pro Asn Asn Leu Phe Met

740 745 750

Val Arg Leu Ala Gln Gly Leu Thr His Leu Gly Lys Gly Thr Leu Thr

755 760 765

Leu Cys Pro Tyr His Ser Asp Arg Gln Leu Met Ser Gln Val Ala Val

770 775 780

Ala Gly Leu Leu Thr Val Leu Val Ser Phe Leu Asp Val Arg Asn Ile

785 790 795 800

Ile Leu Gly Lys Ser His Tyr Val Leu Tyr Gly Leu Val Ala Ala Met

805 810 815

Gln Pro Arg Met Leu Val Thr Phe Asp Glu Glu Leu Arg Pro Leu Pro

820 825 830

Val Ser Val Arg Val Gly Gln Ala Val Asp Val Val Gly Gln Ala Gly

835 840 845

Lys Pro Lys Thr Ile Thr Gly Phe Gln Thr His Thr Thr Pro Val Leu

850 855 860

Leu Ala His Gly Glu Arg Ala Glu Leu Ala Thr Glu Glu Phe Leu Pro

865 870 875 880

Val Thr Pro Ile Leu Glu Gly Phe Val Ile Phe Gly Arg Thr Pro Ile

885 890 895

Met Ile Ser Lys

900

(2) Information SEQ ID NO: 15:

(i) Sequence features:

(A) Length: 995AA

(B) type: amino acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 15:

Lys Lys Met Val Asp Glu Ser Asp Lys Lys Gln Gln Thr Ile Asp Glu

1 5 10 15

Gln Ser Gln Ile Ser Pro Glu Lys Gln Thr Pro Asn Lys Lys Asp Lys

20 25 30

Lys Lys Glu Glu Glu Glu Gln Leu Ser Glu Glu Asp Ala Lys Leu Lys

35 40 45

Thr Asp Leu Glu Leu Leu Val Glu Arg Leu Lys Glu Asp Asp Ser Ser

50 55 60

Leu Tyr Glu Ala Ser Leu Asn Ala Leu Lys Glu Ser Ile Lys Asn Ser

65 70 75 80

Thr Ser Ser Met Thr Ala Val Pro Lys Pro Leu Lys Phe Leu Arg Pro

85 90 95

Thr Tyr Pro Asp Leu Cys Ser Ile Tyr Asp Lys Trp Thr Asp Pro Asn

100 105 110

Leu Lys Ser Ser Leu Ala Asp Val Leu Ser Ile Leu Ala Met Thr Tyr

115 120 125

Ser Glu Asn Gly Lys His Asp Ser Leu Arg Tyr Arg Leu Leu Ser Asp

130 135 140

Val Ser Asp Phe Glu Gly Trp Gly His Glu Tyr Ile Arg His Leu Ala

145 150 155 160

Leu Glu Ile Gly Glu Val Tyr Asn Asp Gln Val Glu Lys Asp Ala Glu

165 170 175

Asp Glu Thr Ser Ser Asp Gly Ser Lys Ser Asp Gly Ser Ala Ala Thr

180 185 190

Ser Gly Phe Glu Phe Ser Lys Glu Asp Thr Leu Arg Leu Cys Leu Asp

195 200 205

Ile Val Pro Tyr Phe Leu Lys His Asn Gly Glu Glu Asp Ala Val Asp

210 215 220

Leu Leu Leu Glu Ile Glu Ser Ile Asp Lys Leu Pro Gln Phe Val Asp

225 230 235 240

Glu Asn Thr Phe Gln Arg Val Cys Gln Tyr Met Val Ala Cys Val Pro

245 250 255

Leu Leu Pro Pro Pro Glu Asp Val Ala Phe Leu Lys Thr Ala Tyr Ser

260 265 270

Ile Tyr Leu Ser Gln Asn Glu Leu Thr Asp Ala Ile Ala Leu Ala Val

275 280 285

Arg Leu Gly Glu Glu Asp Met Ile Arg Ser Val Phe Asp Ala Thr Ser

290 295 300

Asp Pro Val Met His Lys Gln Leu Ala Tyr Ile Leu Ala Ala Gln Lys

305 310 315 320

Thr Ser Phe Glu Tyr Glu Gly Val Gln Asp Ile Ile Gly Asn Gly Lys

325 330 335

Leu Ser Glu His Phe Leu Tyr Leu Ala Lys Glu Leu Asn Leu Thr Gly

340 345 350

Pro Lys Val Pro Glu Asp Ile Tyr Lys Ser His Leu Asp Asn Ser Lys

355 360 365

Ser Val Phe Ser Ser Ala Gly Leu Asp Ser Ala Gln Gln Asn Leu Ala

370 375 380

Ser Ser Phe Val Asn Gly Phe Leu Asn Leu Gly Tyr Cys Asn Asp Lys

385 390 395 400

Leu Ile Val Asp Asn Asp Asn Trp Val Tyr Lys Thr Lys Gly Asp Gly

405 410 415

Met Thr Ser Ala Val Ala Ser Ile Gly Ser Ile Tyr Gln Trp Asn Leu

420 425 430

Asp Gly Leu Gln Gln Leu Asp Lys Tyr Leu Tyr Val Asp Glu Pro Glu

435 440 445

Val Lys Ala Gly Ala Leu Leu Gly Ile Gly Ile Ser Ala Ser Gly Val

450 455 460

His Asp Gly Glu Val Glu Pro Ala Leu Leu Leu Leu Gln Asp Tyr Val

465 470 475 480

Thr Asn Pro Asp Thr Lys Ile Ser Ser Ala Ala Ile Leu Gly Leu Gly

485 490 495

Ile Ala Phe Ala Gly Ser Lys Asn Asp Glu Val Leu Gly Leu Leu Leu

500 505 510

Pro Ile Ala Ala Ser Thr Asp Leu Pro Ile Glu Thr Ala Ala Met Ala

515 520 525

Ser Leu Ala Leu Ala His Val Phe Val Gly Thr Cys Asn Gly Asp Ile

530 535 540

Thr Thr Ser Ile Met Asp Asn Phe Leu Glu Arg Thr Ala Ile Glu Leu

545 550 555 560

Lys Thr Asp Trp Val Arg Phe Leu Ala Leu Ala Leu Gly Ile Leu Tyr

565 570 575

Met Gly Gln Gly Glu Gln Val Asp Asp Val Leu Glu Thr Ile Ser Ala

580 585 590

Ile Glu His Pro Met Thr Ser Ala Ile Glu Val Leu Val Gly Ser Cys

595 600 605

Ala Tyr Thr Gly Thr Gly Asp Val Leu Leu Ile Gln Asp Leu Leu His

610 615 620

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

625 630 635 640

Glu Thr Ala Glu Gly Gln Thr Asn Ser Ile Ser Asp Phe Leu Gly Glu

645 650 655

Gln Val Asn Glu Pro Thr Lys Asn Glu Glu Ala Glu Ile Glu Val Asp

660 665 670

Glu Met Glu Val Asp Ala Glu Gly Glu Glu Val Glu Val Lys Ala Glu

675 680 685

Ile Thr Glu Lys Lys Asn Gly Glu Ser Leu Glu Gly Glu Glu Ile Lys

690 695 700

Ser Glu Glu Lys Lys Gly Lys Ser Ser Asp Lys Asp Ala Thr Thr Asp

705 710 715 720

Gly Lys Asn Asp Asp Glu Glu Glu Glu Lys Glu Ala Gly Ile Val Asp

725 730 735

Glu Leu Ala Tyr Ala Val Leu Gly Ile Ala Leu Ile Ala Leu Gly Glu

740 745 750

Asp Ile Gly Lys Glu Met Ser Leu Arg His Phe Gly His Leu Met His

755 760 765

Tyr Gly Asn Glu His Ile Arg Arg Met Val Pro Leu Ala Met Gly Ile

770 775 780

Val Ser Val Ser Asp Pro Gln Met Lys Val Phe Asp Thr Leu Thr Arg

785 790 795 800

Phe Ser His Asp Ala Asp Leu Glu Val Ser Met Asn Ser Ile Phe Ala

805 810 815

Met Gly Leu Cys Gly Ala Gly Thr Asn Asn Ala Arg Leu Ala Gln Leu

820 825 830

Leu Arg Gln Leu Ala Ser Tyr Tyr Ser Arg Glu Gln Asp Ala Leu Phe

835 840 845

Ile Thr Arg Leu Ala Gln Gly Leu Leu His Leu Gly Lys Gly Thr Met

850 855 860

Thr Met Asp Val Phe Asn Asp Ala His Val Leu Asn Lys Val Thr Leu

865 870 875 880

Ala Ser Ile Leu Thr Thr Ala Val Gly Leu Val Ser Pro Ser Phe Met

885 890 895

Leu Lys His His Gln Leu Phe Tyr Met Leu Asn Ala Gly Ile Arg Pro

900 905 910

Lys Phe Ile Leu Ala Leu Asn Asp Glu Gly Glu Pro Ile Lys Val Asn

915 920 925

Val Arg Val Gly Gln Ala Val Glu Thr Val Gly Gln Ala Gly Arg Pro

930 935 940

Lys Lys Ile Thr Gly Trp Ile Thr Gln Ser Thr Pro Val Leu Leu Asn

945 950 955 960

His Gly Glu Arg Ala Glu Leu Glu Thr Asp Glu Tyr Ile Ser Tyr Thr

965 970 975

Ser His Ile Glu Gly Val Val Ile Leu Lys Lys Asn Pro Asp Tyr Arg

980 985 990

Glu Glu Glu

995

(2) Information SEQ ID NO: 16:

(i) Sequence features:

(A) Length: 945AA

(B) type: amino acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: protein

(xi) Sequence description: SEQ ID NO: 16:

Met Ser Leu Thr Thr Ala Ala Pro Leu Leu Ala Leu Leu Arg Glu Asn

1 5 10 15

Gln Asp Ser Val Lys Thr Tyr Ala Leu Glu Ser Ile Asn Asn Val Val

20 25 30

Asp Gln Leu Trp Ser Glu Ile Ser Asn Glu Leu Pro Asp Ile Glu Ala

35 40 45

Leu Tyr Asp Asp Asp Thr Phe Ser Asp Arg Glu Met Ala Ala Leu Ile

50 55 60

Ala Ser Lys Val Tyr Tyr Asn Leu Gly Glu Tyr Glu Ser Ala Val Lys

65 70 75 80

Tyr Ala Leu Ala Ala Lys Asp Arg Phe Asp Ile Asp Glu Lys Ser Gln

85 90 95

Phe Val Glu Thr Ile Val Ser Lys Ser Ile Glu Met Tyr Val Gln Glu

100 105 110

Ala Ser Lys Gln Tyr Thr Lys Asp Glu Gln Phe Tyr Thr Lys Asp Ile

115 120 125

Ile Asp Pro Lys Leu Thr Ser Ile Phe Glu Arg Met Ile Glu Lys Cys

130 135 140

Leu Lys Ala Ser Glu Leu Lys Leu Ala Leu Gly Ile Ala Leu Glu Gly

145 150 155 160

Tyr Arg Leu Asp Ile Ile Glu Ser Ala Leu Lys Ser Lys Leu Asp Gln

165 170 175

Asp Ser Thr Ser Glu Asn Val Lys Ile Ile Asn Tyr Leu Leu Thr Leu

180 185 190

Ala Ile Thr Thr Val Thr Asn Ser Lys Phe Arg Ser Ser Ile Leu Arg

195 200 205

Lys Ser Phe Asp Phe Leu Met Asn Met Pro Asn Cys Asp Tyr Leu Thr

210 215 220

Leu Asn Lys Val Val Val Asn Leu Asn Asp Ala Gly Leu Ala Leu Gln

225 230 235 240

Leu Phe Lys Lys Leu Lys Glu Glu Asn Asp Glu Gly Leu Ser Ala Gln

245 250 255

Ile Ala Phe Asp Leu Val Ser Ser Ala Ser Gln Gln Leu Leu Glu Ile

260 265 270

Leu Val Thr Glu Leu Thr Ala Gln Gly Tyr Asp Pro Ala Leu Leu Asn

275 280 285

Ile Leu Ser Gly Leu Pro Thr Cys Asp Tyr Tyr Asn Thr Phe Leu Leu

290 295 300

Asn Asn Lys Asn Ile Asp Ile Gly Leu Leu Asn Lys Ser Lys Ser Ser

305 310 315 320

Leu Asp Gly Lys Phe Ser Leu Phe His Thr Ala Val Arg Leu Ala Asn

325 330 335

Gly Phe Met His Ala Gly Thr Thr Asp Asn Ser Phe Ile Lys Ala Asn

340 345 350

Leu Pro Trp Leu Gly Lys Ala Gln Asn Trp Ala Lys Phe Thr Ala Thr

355 360 365

Ala Ser Leu Gly Val Ile His Lys Gly Asn Leu Leu Glu Gly Lys Lys

370 375 380

Val Met Ala Pro Tyr Leu Pro Gly Ser Arg Ala Ser Ser Arg Phe Ile

385 390 395 400

Lys Gly Gly Ser Leu Tyr Gly Leu Gly Leu Ile Tyr Ala Gly Phe Gly

405 410 415

Arg Asp Thr Thr Asp Tyr Leu Lys Asn Ile Ile Val Glu Asn Ser Gly

420 425 430

Thr Ser Gly Asp Glu Asp Val Asp Val Leu Leu His Gly Ala Ser Leu

435 440 445

Gly Ile Gly Leu Ala Ala Met Gly Ser Ala Asn Ile Glu Val Tyr Glu

450 455 460

Ala Leu Lys Glu Val Leu Tyr Asn Asp Ser Ala Thr Ser Gly Glu Ala

465 470 475 480

Ala Ala Leu Gly Met Gly Leu Cys Met Leu Gly Thr Gly Lys Pro Glu

485 490 495

Ala Ile His Asp Met Phe Thr Tyr Ser Gln Glu Thr Gln His Gly Asn

500 505 510

Ile Thr Arg Gly Leu Ala Val Gly Leu Ala Leu Ile Asn Tyr Gly Arg

515 520 525

Gln Glu Leu Ala Asp Asp Leu Ile Thr Lys Met Leu Ala Ser Asp Glu

530 535 540

Ser Leu Leu Arg Tyr Gly Gly Ala Phe Thr Ile Ala Leu Ala Tyr Ala

545 550 555 560

Gly Thr Gly Asn Asn Ser Ala Val Lys Arg Leu Leu His Val Ala Val

565 570 575

Ser Asp Ser Asn Asp Asp Val Arg Arg Ala Ala Val Ile Ala Leu Gly

580 585 590

Phe Val Leu Leu Arg Asp Tyr Thr Thr Val Pro Arg Ile Val Gln Leu

595 600 605

Leu Ser Lys Ser His Asn Ala His Val Arg Cys Gly Thr Ala Phe Ala

610 615 620

Leu Gly Ile Ala Cys Ala Gly Lys Gly Leu Gln Ser Ala Ile Asp Val

625 630 635 640

Leu Asp Pro Leu Thr Lys Asp Pro Val Asp Phe Val Arg Gln Ala Ala

645 650 655

Met Ile Ala Leu Ser Met Ile Leu Ile Gln Gln Thr Glu Lys Leu Asn

660 665 670

Pro Gln Val Ala Asp Ile Asn Lys Asn Phe Leu Ser Val Ile Thr Asn

675 680 685

Lys His Gln Glu Gly Leu Ala Lys Phe Gly Ala Cys Val Ala Gln Gly

690 695 700

Ile Met Asn Ala Gly Gly Arg Asn Val Thr Ile Gln Leu Glu Asn Ala

705 710 715 720

Asp Thr Gly Thr Leu Asp Thr Lys Ser Val Val Gly Leu Val Met Phe

725 730 735

Ser Gln Phe Trp Tyr Trp Phe Pro Leu Ala His Phe Leu Ser Leu Ser

740 745 750

Phe Thr Pro Thr Thr Val Ile Gly Ile Arg Gly Ser Asp Gln Ala Ile

755 760 765

Pro Lys Phe Gln Met Asn Cys Tyr Ala Lys Glu Asp Ala Phe Ser Tyr

770 775 780

Pro Arg Met Tyr Glu Glu Ala Ser Gly Lys Glu Val Glu Lys Val Ala

785 790 795 800

Thr Ala Val Leu Ser Thr Thr Ala Arg Ala Lys Ala Arg Ala Lys Lys

805 810 815

Thr Lys Lys Glu Lys Gly Pro Asn Glu Glu Glu Lys Lys Lys Glu His

820 825 830

Glu Glu Lys Glu Lys Glu Arg Glu Thr Asn Lys Lys Gly Ile Lys Glu

835 840 845

Thr Lys Glu Asn Asp Glu Glu Phe Tyr Lys Asn Lys Tyr Ser Ser Lys

850 855 860

Pre Tyr Lys Val Asp Asn Met Thr Arg Ile Leu Pro Gln Gln Ser Arg

865 870 875 880

Tyr Ile Ser Phe Ile Lys Asp Asp Arg Phe Val Pro Val Arg Lys Phe

885 890 895

Lys Gly Asn Asn Gly Val Val Val Leu Arg Asp Arg Glu Pro Lys Glu

900 905 910

Pro Val Ala Leu Ile Glu Thr Val Arg Gln Met Lys Asp Val Asn Ala

915 920 925

Pro Leu Pro Thr Pro Phe Lys Val Asp Asp Asn Val Asp Phe Pro Ser

930 935 940

Ala

945

(2) Information SEQ ID NO: 17:

(i) Sequence features:

(A) Length: 142AA

(B) type: amino acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 17:

Trp Xaa Ile Arg Ser Asp Glu Arg Val Leu Gln Tyr Gly Glu Gln Asn

1 5 10 15

Ile Arg Arg Ala Val Pro Leu Ala Leu Gly Leu Leu Cys Ile Ser Asn

20 25 30

Pro Lys Val Thr Val Met Asp Thr Leu Ser Arg Leu Ser His Asp Arg

35 40 45

Phe Arg Ser Cys Asn Gly Ser Asn Tyr Leu Pro Trp Ile Asp Arg Arg

50 55 60

Trp Asn Gln Gln Cys Lys Asp Ser Trp His Ala Lys Ser Leu Gln Leu

65 70 75 80

Leu Leu Gln Gly Cys Pro Xaa Phe Phe Ser Val Cys Ala Ser Leu Lys

85 90 95

Gly Phe Xaa His Met Gly Lys Gly Leu Leu Thr Leu Asn Pro Phe His

100 105 110

Ser Glu Arg Ala Xaa Phe Leu Xaa Xaa Asn Pro Asp Phe Pro Trp Val

115 120 125

Gly Xaa Asn Phe Leu Gln Xaa Xaa Xaa Phe Xaa Ile Glu Thr

130 135 140

(2) Information SEQ ID NO: 18:

(i) Sequence features:

(A) Length: 97AA

(B) type: amino acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: peptide

(xi) Sequence description: SEQ ID NO: 18:

Met Gln Pro Arg Met Leu Thr Thr Leu Val Glu Asp Glu Met Lys Pro

1 5 10 15

Gly Ser Leu Lys Gln Leu Asn Val Ser Val Arg Val Gly Gln Pro Val

20 25 30

Asp Val Val Ala Gln Ala Gly Lys Pro Lys Thr Ile Thr Gly Phe Gln

35 40 45

Thr His Thr Thr Pro Val Leu Leu Ala His Gly Glu Arg Ala Glu Leu

50 55 60

Ala Asn Asp Glu Tyr Leu Ser Val Thr Pro His Leu Glu Gly Leu Val

65 70 75 80

Ile Leu Lys Lys Asn Pro Asp Tyr Gln Pro Val Val Val Ser Thr Lys

85 90 95

Lys

(2) Information SEQ ID NO: 19:

(i) Sequence features:

(A) Length: 390bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 19:

ATCAGTGTCA CTACGGATAG TGATGACACT CACAGGAGGG CTGGGGGTAT CTGGAATGAT 60

GATTGGCTGA TGCTGGTCTT GGACAGGAAC TAGGGAATTA TAAGAAGATG TGGTACGAAG 120

AGGACTACTC CCANCCAGAG AATAAACTTG AGAAGGCAGG ACTTCCAGAG AGGATTTGGA 180

TGAAACTGGA GCAGACTGCT TATTCTACTT TGAAGGGAGG GAACTAGACT GTTGTTGTCT 240

GACAACATGG GCAACACCAA CATTCAGAGG CTGAGCAGTN GCCAAGGNCA CATGGTTGGT 300

CAGCAAAGAT GGCTGCTGCA TAATAGTGCT GTACTGGTCG NCATGAGAGT GGGCATTCCC 360

CAGTCAGCTA GCTGGTGGGC TGCTCCCCAT 390

(2) Information SEQ ID NO: 20:

(i) Sequence features:

(A) Length: 385bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 20:

CCTCTCAGTT ATCTCTGTTG GAGTAGTCCT CTTCGTACCA CATCTTCTTA TAATTCCCTA 60

GTTCCTGTCC AAGACCAGCA TCAGCCAATC ATCATTCCAG ATACCCCCAG CCCTCCTGTG 120

AGTGTCATCA CTATCCGTAG TGACACTGAT GAAGAAGAGG ACAACAAATA CAAGCCCAAT 180

AGCTCGAGCC TGAAGGCGAG GTCTAATGTC ATCAGTTATG TCACTGTCAA TGATTCTCCA 240

GACTCTGACT CCTCCCTGAG CAGCCCACAT TCCACAGCCA CTCTGAGTGC TCTGCGGGGC 300

AACAGTGGGA CCCTTCTGGA GGGACCTGGC AGACCTGCAG CAGATGGCAT TGGCACCCGT 360

ACTATCATTG TACCTGAGCG GCCGC 385

(2) Information SEQ ID NO: 21:

(i) Sequence features:

(A) Length: 444bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 21:

GGGAGCCTGT GCACCCCGAT GTCACCATGA AGCCACTGCC CTTCTATGAA GTCTATGGGG 60

AGCTCATCCG ACCCACCACC CTTGCGTCCA CCTCCAGCCA GAGGTTCGAG GAAGCCCACT 120

TCACCTTCGC GCTCACTCCC CAGCAGCTGC AGCAGATTCT CACGTCCAGG GAGGTTATGC 180

CAGGAGCCAA GTGTGATTAC ACCATACAAG TGCAGCTCAG ATTCTGTCTC TGTGAGACCA 240

GCTGCCCTCA GGAGGACTAT TTCCCCCCTA ACCTCTTTGT TAAGGTTAAT GGGAAACTCT 300

GCCCCCTGCC GGGTTACCTC CCTCCAACCC AAGAATGGAG CTGAGCCCAA GAGGCCCAGC 360

CGTCCGATCA ACATCACACC CTTGGCTCGA CTCTCAGCCA CTGTCCCCAA CACCATCGTA 420

GTTAATTGGG TCATCTTGAA GTTT 444

(2) Information SEQ ID NO: 22:

(i) Sequence features:

(A) Length: 888bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 22:

TCCAACACCA TCGTAGATAA ATTGGTCATC TGAGTTTGGA CCGGAATTAC TCCTTGTCCG 60

TGTACCCTGG TGAGGCAATT GACTGCAGGG ACCCTTCTAC ACAAACTCAG AGCCAAGGGG 120

ATCCGGAATC CAGACCATTC CCGGGCACTG ATCAAGGAGA AACTGACTGC TGACCCCGAC 180

AGTGAAGTGG CTACTACAAG TCTCCGGGTG TCACTCATGT GCCCGCTAGG GAAGATGCGC 240

CTGACTGTCC CGTGTCGTGC CCTCACCTGT GCCCATCTGC AGAGTTTCGA TGCTGCCCTT 300

TATCTACAGA TGAATGAGAA GAAGCCGACA TGGACGTGTC CTGTGCGTGA CAAGAAGGCT 360

CCCTATGAGT CGCTGATTAT TGATGGTTTA TTCATGGAAA TTCTTAATTC CTGTTCGGAT 420

TGTGATGAGA TCCAGTTCAT GGAAGATGGA TCCTGGTGTC CGATGAAACC CAAGAAGGAG 480

GCATCAGAGG TTTGCCCCCC GCCAGGGTAT GGGCTGGATG GTTCCAGTA CAGCGCAGTC 540

CAGGAGGGAA TTCAGCAGA GAGTAAGAAG AGGGTCGAAG TCATTGACTT GACCATCGAA 600

AGCTCATCAG ATGAGGAGGA TTTGCCCCCC ACCAAGAAGC ACTGCCCTGT CACCCTCAGCG 660

GTCATTCCAG CCCTTCCTGG AAGCAAAGGA GCCCTGACCT CTGGTCACCA GCCATCCTCG 720

GTGCTGCGGA GCCCTGCAAT GGGCACACTG GGCAGTGACT TCCTGTCTAG TCTCCCGCTA 780

CATGAGTACC CACCTGCCTT CCCACTGGGG GTTGACATCC AAGGTTTAGA TTTTATTTTC 840

TTTTCTTCAG ACTGAGAGTC AGAATTACGG GCCTTCAGTT ATCATTCG 888

(2) Information SEQ ID NO: 23:

(i) Sequence features:

(A) Length: 392bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 23:

CCACTTCCTG GCCCACTGCC CCCAAACTGG GGACTTCTCAC CGCAAGCTCC AACTCCAGCG 60

CCCCCTCCTG GTCGTGTCAG CAGCATTGTG GCTCCTGGGA GCTCCTTGAG GGAAGGGCAT 120

GGAGGACCCC TGCCTTCAGG TCCCTCTTTG ACTGGCTGTC GGTCAGACGT CATTTCCTTG 180

GACTGAGCTT TTTGGATTAT GAATCAATC TCCATTGGCC CCAGCACTGA GCAGATCACG 240

TTGTGGGTTC CGAACCCCTG GCTGCTCTGA TCCCTCAGGG GTCATTGGCC AAAGGCCAGG 300

CCAGAGCTTC ATGGATACCT GCTTTTGGCC TTATCGCTGC CTAACAGGCC AGTACTCACA 360

GGGTTAACAT TTAACCTTTT TATGGTGGCC CG 392

(2) Information SEQ ID NO: 24:

(i) Sequence features:

(A) Length: 425bp

(B) type: nucleic acid

(C) Chain type: single chain

(D) Topology: Linear

(ii) Molecular type: cDNA

(xi) Sequence description: SEQ ID NO: 24:

AATTCGGCAC GAGGTTGTGC TGTGGGGAAG GGAGAAGGAT TTGTAAACCC CGGAGCGAGG 60

TTCTGCTTAC CCGAGGCCGC TGCTGTGCGG AGACCCCCGG GTGAAGCCAC CGTCATCATG 120

TCTGACCAGG AGGCAAAACC TTCCAACTGA GGACTTGGGG GATAAGAAGG AAGGTGAATA 180

TATTAAACTC AAAGTCATTG GACAGGATAG CAGTGAGATT CACTTCAAAG TGAAAATGAC 240

AACACATCTC AAGAAACTCA AAGAATCATA CTGTCAAAGA CAGGGTGTTC CAATGAATTC 300

ACTCAGGTTT CTCTTTGAGG GTCAGAGAAT TGCTGATAAT CATACTCCAA AAGAACTGGG 360

AATGGAGAAG AAAGATTGTG ATTTGAAGTT TTATCAGGAA CAAACGGGGG GTCATTCAAC 420

AGCTT 425

(2) Information SEQ ID NO: 25:

(i) Sequence features:

(A) Length: 455AA

(B) type: amino acid

(C) Chain type: single chain

(D) Topology Linear

(ii) Molecular type: polypeptide

(xi) Sequence description: SEQ ID NO: 25:

mglstvpdll lplvllellv giypsgvigl vphlgdrekr dsvcpqgkyi hpqnnsicct 60

kchkgtylyn dcpgpgqdtd crecesgsft asenhlrhel seskcrkemg qveissctvd 120

rdtvcgcrkn qyrhywsenl fqcfncslcl ngtvhlscqe kqntvctcha gfflrenecv 180

scsnckksle ctklclpqie nvkgtedsgt tvllplviff glcllsllfi glmyryqrwk 240

sklysivcgk stpekegele gtttkplapn psfsptpgft ptlgfspvps stftssstyt 300

pgdcpnfaap rrevappyqg adpilatala sdpipnplqk wedsahkpqs ldtddpatly 360

avvenvpplr wkefvrrlgl sdheidrlel qngrclreaq ysmlatwrrr tprreatlel 420

lgrvlrdmdl lgclediea lcgpaalppa psllr 455

(2) Information SEQ ID NO: 26:

(i) Sequence features:

(A) Length: 454AA

(B) type: amino acid

(C) Chain type: single chain

(D) Topology Linear

(ii) Molecular type: polypeptide

(xi) Sequence description: SEQ ID NO: 26:

mglptvpgll lslvllallm gihpsgvtgl vpslgdrekr dslcpqgkyv hsknnsicct 60

kchkgtylvs dcpspgrdtv crecekgtft asqnylrqcl scktcrkems qveispcqad 120

kdtvcgcken qfqrylseth fqcvdcspcf ngtvtipcke tqntvcncha gfflresecv 180

pcshckknee cmklclpppl anvtnpqdsg tavllplvil lglcllsfif islmcryprw 240

rpevysiicr dpvpvkeeka gkpltpapsp afsptsgfnp tlgfstpgfs spvsstpisp 300

ifgpsnwhfm ppvsevvptq gadpllyesl csvpaptsvq kwedsahpqr pdnadlaily 360

avvdgvppar wkefmrfmgl seheierlem qngrclreaq ysmleawrrr tprhedtlev 420

vglvlskmnl agclenilea lrnpapsstt rlpr 454

Claims (43)

1. dna sequence dna, a kind of protein that can be incorporated into the intracellular region of an acceptor in tumour necrosis factor/nerve growth factor (TNF/NGF) receptor superfamily of encoding.

2. a kind of dna sequence dna as claimed in claim 1, wherein said acceptor are to be selected from a same clan that is made up of P75TNF-R and FAS aglucon acceptor.

3. a kind of dna sequence dna as claimed in claim 1 is selected from a DNA same clan, is made up of following each sequence:

(a) a kind of cDNA sequence is derived from the coding region of natural TNF-R or FAS-R intracellular region-conjugated protein;

(b) some dna sequence dnas can be hybridized the sequence in (a) under mild conditions, and the TNF-R of coding biologically active or intracellular region-conjugated protein of FAS-R; And

(c) some dna sequence dnas, they since the degeneracy of genetic code and degeneracy become (a) and (b) in defined dna sequence dna, their the encode TNF-R of biologically active or intracellular region-conjugated proteins of FAS-R.

4. a kind of dna sequence dna as claimed in claim 1, coding P75TNF-R intracellular region (P75IC)-conjugated protein.

5. a kind of dna sequence dna as claimed in claim 4, a kind of protein of encoding is selected from an albumen same clan, and this same clan includes specified protein 75.3 and 75.16 herein.

6. a kind of dna sequence dna as claimed in claim 1, a kind of FAS-R intracellular region (the FAS-IC)-conjugated protein of encoding.

7. a kind of dna sequence dna as claimed in claim 6, a kind of protein of encoding is selected from an albumen same clan, and this same clan comprises protein F2, F9 and the DD11 that this paper is specified.

8. a kind of dna sequence dna as claimed in claim 7, among coded protein F2, F9 and the DD11 any one, include the same clan that a kind of dna sequence dna is selected from following DNA: SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23 and SEQ ID NO:24.

9. a carrier includes the described dna sequence dna of claim 1.

More 10. the eucaryon or the prokaryotic host cell that have transformed contain the described carrier of claim 9.

11. method of producing protein, analogue or derivative, these protein, analogue or derivative can be incorporated into the intracellular region of a kind of acceptor that belongs to tumour necrosis factor/nerve growth factor (TNF/NGF) superfamily, and this method comprises: cultivate the described transformed host cell of claim 10 under the condition that is fit to expression said protein, analogue or derivative; Carry out proteinic posttranslational modification so that obtain said protein, and extract said marking protein, analogue or derivative in the substratum of said transformant or in the cell extract of said transformant.

12. a protein, or its analogue and derivative, by any one described dna sequence encoding among the claim 1-8, perhaps this protein, its analogue and derivative can be incorporated into the intracellular region of TNF or FAS acceptor.

13. as claimed in claim 12 a kind of protein-based, be selected from an albumen same clan, this family includes protein 75 .3, and 75.16, F2, F9 and DD11, and analogue of their biologically actives and derivative.

14. some antibody, or its active fragments or derivatives thereof, for the described albumen of claim 12, its analogue or derivative have specificity.

15. a method is used to regulate the effect of TNF or FAS-R ligand pair cell, these cells are loaded with TNF-R or FAS-R, this method comprises with the described antibody of claim 14, or its active fragments or derivatives thereof is handled said cell, said processing is to contain said antibody, the suitable ingredients of its active fragments or derivative is applied to said cell, wherein when the IC-of said cell conjugated protein is exposed to born of the same parents' outside surface, then said component is just prepared as born of the same parents and is used outward, and when said IC-conjugated protein be in the born of the same parents time, then said component is just prepared as using in the born of the same parents.

16. a method is used to regulate TNF or the FAS-R aglucon effect to the cell that is loaded with TNF-R or FAS-R, comprise and use one or more protein, analogue or derivative are handled said cell, said protein, analogue or derivative are to be selected from an albumen same clan, this same clan comprises the more described protein of claim 12, analogue and derivative, and a kind of protein, be FAS-IC or FAS-DD, its analogue or derivative, the activity that all these protein can both be incorporated into intracellular region and regulate said TNF-R or FAS-R, wherein said cell is handled and is comprised: with one or more protein, analogue or derivative are introduced in the said cell to be applicable to the form of introducing in its born of the same parents, one or more protein of perhaps will encoding, the dna sequence dna of analogue or derivative is introduced in the said cell with the suitable carrier format that is loaded with this sequence, and carrier is said sequence can be gone in the said cell with the mode intercalation of expressing.

17. a method is used to regulate TNF or the FAS-R ligand effect to the cell that carries TNF-R or FAS-R, comprise with the said cell of an oligonucleotide series processing, this nucleotide sequence is the antisense sequences that is selected from least a portion of the described sequence of coding claim 1, and the antisense sequences that is selected from coding FAS-IC or FAS-DD encoding sequence, this oligonucleotide sequence can be sealed at least a expression in the intracellular region conjugated protein of TNF-R or FAS-R.

18. a method that is used to handle tumour cell or HIV cells infected or other ill cell comprises:

(a) make up a kind of recombinant animal virus vector, be loaded with a kind of encoding sequence of virus capsid protein, the acceptor that this albumen can be incorporated into a specific tumors cell surface receptor or HIV-cells infected surface receptor or be carried by other sick cell, this carrier also has an encoding sequence, a kind of protein of coding is selected from some protein, analogue and derivative, they can be incorporated into one or more intracellular regions of one or more acceptors of tumour necrosis factor/nerve growth factor (TNF/NGF) receptor superfamily, the intracellular region of FAS-R (FAS-IC), or its " dead functional domain " (FAS-DD), the perhaps analogue of its biologically active or derivative, said protein is when being expressed in the said tumour cell, just can kill these cells in the time of in the IHV-cells infected or in other ill cell; And

(b) infect said tumour cell or HIV-cells infected or other ill cell with said carrier in (a).

19. method that is used to regulate TNF or AFS-R ligand pair cell effect, comprise the ribozyme technology of using, wherein the carrier of encoding ribozyme sequence can interact with the described a kind of proteinic cell mRNA sequence of coding claim 12 or interact with the mRNA sequence of coding FAS-IC or FAS-DD, this carrier medicament is introduced in the said cell in the mode that is fit to the ribozyme sequence expression, when said ribozyme sequence is expressed in the said cell, it just interacts with said cell mRNA sequence and cuts said mRNA sequence, and the result has just suppressed said protein or said FAS-IC or the expression of FAS-DD in said born of the same parents.

20. the method for separation and identification of protein, it is conjugated protein that these protein can be incorporated into the described intracellular region of claim 12, this method comprises the two hybrid technology of using yeast, a kind of sequence of encoding said intracellular region conjugated protein is to be carried by a hybrid carrier (hybrid vector) in this technology, another sequence from cDNA or gene DNA gene pool is to be carried by the second hybrid carrier (second hybrid vector), then these carriers are used for the transformed yeast host cell and separate positivity (positive) transformant, extract the said second hybrid carrier subsequently and a kind of proteic sequence that obtains encoding, this albumen can be incorporated into said intracellular region conjugated protein.

21. medicinal compositions that is used to regulate TNF or FAS-R ligand pair cell effect, include as claimed in claim 12 a kind of protein, perhaps protein FAS-R or FAS-DD, the fragment of their biologically actives as activeconstituents, analogue, derivative or their mixture.

22. medicinal compositions that is used to regulate the effect of TNF or FAS-R part pair cell, include a kind of recombinant animal virus vector as activeconstituents, a kind of protein of this vector encoded can be in conjunction with cell surface receptor, this carrier described a kind of protein of claim 12 of also encoding, or protein FAS-IC or FAS-DD, the fragment of its biologically active or analogue.

23. a medicinal compositions that is used to regulate TNF or FAS-R aglucon pair cell effect includes a kind of oligonucleotide sequence as activeconstituents, the antisense sequences of the described sequence of this sequence encoding claim 1.

A 24. soluble oligomeric Tumor Necrosis Factor Receptors (TNF-R), include at least two kinds from-association fused protein, each fused protein has: (a) at the one end, one TNF land, be selected from extracellular region, its analogue or derivatives thereof of TNF-R, said extracellular region, its analogue or derivatives thereof are can not produce deleterious causing from-association the TNF bonded is disturbed, but can be in conjunction with TNF; And (b) at its another end, one is selected from all intracellular regions (P55IC) that (i) comes down to P55TNF-R from the-district that associates, from about amino-acid residue the 206th of natural P55TNF-R molecule to the section about amino-acid residue the 426th; The (ii) dead functional domain of P55IC, from about amino-acid residue the 328th of natural P55TNF-R molecule to the section about amino-acid residue the 426th; (iii) come down to all intracellular regions (Fas-IC) of Fas/APOl acceptor; The (iv) dead functional domain of Fas-IC; And (V) (i)-(iv) can produce analogue, fragment or derivative from-association in any one, wherein said at least two kinds only produce from-association at more said ends (b) from-Rapsyn matter, and more said ends (a) that it has can be in conjunction with a TNF monomer; And the salt and the functional deriv that include said solubility oligopolymer TNF-R.

25. a kind of soluble oligomeric TNF-R as claimed in claim 24, include: as said at least two ends (a), essence is all extracellular regions of P55-R, from about amino-acid residue 1 of P55TNF-R molecule to the amino acid section between about amino-acid residue 172, and, come down to all said dead functional domains of P55-IC as its at least two ends (b).

26. a kind of soluble oligomeric TNF-R as claimed in claim 24, include: as its two ends (a), the analogue of the intracellular region of P55-R or derivative, each said analogue or derivative can be in conjunction with TNF monomers, but can not produce from-association; And, come down to all said dead functional domains of P55-IC as its at least two ends (b).

27. a kind of soluble oligomeric TNF-R as claimed in claim 24, include: as said at least two ends (a), come down to all extracellular regions of P55-R, from about amino-acid residue 1 of P55TNF-R molecule to the section about amino-acid residue 172, and, come down to all said dead functional domains of Fas-IC as its at least two ends (b).

28. a kind of soluble oligomeric TNF-R as claimed in claim 24, include: as its analogue or the derivative of extracellular region of P55-R of at least two ends (a), each said analogue or derivative all can be in conjunction with TNF monomers, but can not take place, and as its all said dead functional domains that come down to Fas-IC of at least two ends (b) from-association.

29. an expression vector includes a fused protein encoding sequence, the said fused protein of coding claim 24.

30. a host cell contains the described a kind of carrier of claim 29, and can express said fused protein encoding sequence.

31. a pharmaceutical composition includes soluble oligomeric TNF-R as claimed in claim 24, its esters or its functional deriv, and a kind of pharmaceutically acceptable carrier.

32. the method for the deleterious effect of an antagonism TNF promptly produces or the external treatment process that causes under the excessive situation of TNF of using in owing to body, this method comprises to be used the described pharmaceutical composition of claim 31 to needed curee.

33. a method is used to keep TNF persistent advantageous effect in mammalian body, comprising combines the described pharmaceutical composition of claim 31 with TNF carries out external using.

A 34. solubility oligopolymer Fas/APOl acceptor (Fas-R), include at least two kinds from-association fused protein, every kind of fused protein has (a) at they ends, one Fas ligand-binding domain, be selected from the extracellular region of Fas-R, its analogue or derivatives thereof can not take place from-association but can be in conjunction with the Fas aglucon; (b) at its another end, one is selected from from-the district that associates: (i) come down to all intracellular regions (P55-IC) of P55TNF-R, from about amino-acid residue the 206th of P55TNF-R molecule to the section about amino-acid residue 426; The (ii) dead functional domain of P55-IC, from about the 328th amino-acid residue of P55TNF-R molecule to the section about amino-acid residue 426; (iii) be essentially all intracellular regions (Fas-IC) of Fas/APOl acceptor; The (iv) dead functional domain of Fas-IC; And (v) (i)-(iv) any can produce analogue or the derivative from-association, wherein said at least two kinds only produce from-association at said end (b) from-Rapsyn matter, more said ends (a) that it has can be in conjunction with at least two Fas aglucon monomers, and each end (a) can be in conjunction with a Fas aglucon monomer; And the salt and the functional deriv that contain said soluble oligomeric Fas-R.

35. an expression vector includes a fusion rotein encoding sequence, the more said fusion roteins of coding claim 34.

36. a host cell contains the described a kind of carrier of claim 35, and can express said fused protein sequence.

37. a pharmaceutical composition includes the described soluble oligomeric Fas-R as activeconstituents of claim 34, its esters or its functional deriv or its mixture, and a kind of medicinal acceptable carrier of going up.

38. the method for an antagonism Fas aglucon deleterious effect, promptly since body in the spontaneous or external treatment process that causes under the excessive situation of FAS aglucon of using.This method comprises uses the curee of the described medicinal component of claim 37 to this medicine of needs.

39. soluble oligomeric acceptor (mixing the affinity acceptor) that simultaneously TNF and FAS-R aglucon is had affinity, include at least two kinds from-association fused protein, one of this fused protein is to come from the described TNF-R of claim 24, and TNF is had specific protein; Another kind of fused protein is a kind of oligomerization Fas/APOl acceptor (FAS-R) of solubility, include at least two kinds from-association fused protein, every kind of fused protein has (a) end in it, one Fas ligand-binding domain, be selected from the extracellular region of Fas-R, or its analogue or derivatives thereof, all can not produce from-association but can be in conjunction with the Fas aglucon; And (b) at its other end, one is selected from all intracellular regions (P55-IC) that (i) comes down to TNF-R from the-district that associates, from the amino-acid residue of P55TNF-R molecule the about the 206th to amino-acid residue the 426th section; The (ii) dead functional domain of P55-IC, the section from the amino-acid residue of P55TNF-R molecule the about the 328th to amino-acid residue the about the 426th; (iii) come down to all intracellular regions (Fas-IC) of Fas/APOl acceptor; The (iv) dead functional domain of Fas-IC; And (the v) described analogue or the derivative that can produce self-association of any one of (i)-(iv), wherein said at least two kinds from-Rapsyn matter only can more said ends (b) produce from-associate, more said ends (a) that it has can be in conjunction with at least two kinds of Fas aglucon monomers, and each end (a) can be in conjunction with a Fas aglucon monomer; And some salts and the functional deriv that include said soluble oligomeric Fas-R.

40. a pharmaceutical composition includes claim 39 described mixing affinity acceptor and a kind of medicinal acceptable carrier of going up as activeconstituents.

41. the method for antagonism TNF and FAS-R aglucon deleterious effect comprises and uses the described pharmaceutical composition of claim 40.

42. a kind of pharmaceutical composition as claimed in claim 21 by the TNF correlation effect in the inducing cell, and is used to handle cell, include P55-IC as activeconstituents, its some fragment, its analogue and derivative thereof, and a kind of medicinal acceptable carrier of going up.

43. a method of handling tumour comprises that using the described medicinal component of claim 42 expresses also kill tumor cell subsequently to bring out IL-8.

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