RU2241747C2 - Dna molecule encoding polypeptide able to bind with intracellular domain of fas (fas-ic) ligand receptor, polypeptide, method for its preparing, vector, method for modulation of effect of fas-r ligand on cells (variants), pharmaceutical composition (variants), method for isolation and identification of protein - Google Patents

Abstract

FIELD: biotechnology, genetic engineering, medicine, pharmacy.

SUBSTANCE: polypeptide able to bind with intracellular domain of Fas-R is prepared by culturing cells transformed with vector comprising the DNA sequence encoding indicated polypeptide. The modulation of effect of Fas-R ligand is carried out using polypeptide or mRNA encoding polypeptide. Pharmaceutical composition used for modulation of effect of Fas-R ligand on cells comprises indicated polypeptide or viral vector encoding its. Invention provides carrying out the development of therapeutic agents for modulation of cytotoxic activity. Invention can be used for preparing protein able to modulate function of Fas-receptor.

EFFECT: improved isolating and identifying method, valuable biological and medicinal properties of DNA, polypeptide and composition.

12 cl, 11 dwg, 1 ex

Description

Field of Invention

This invention generally relates to the field of receptors belonging to the superfamily of TNF / NGF receptors and the control of their biological functions. The TNF / NGF receptor family includes receptors such as p55 and p75 tumor necrosis factor (TNF-R) and a FAS ligand receptor (also called FAS / APOI or FAS-R, hereinafter referred to as FAS-R) and others. Specifically, the present invention relates to a new protein, designated here MORT-I (also called HF-I), which binds to the intracellular domain (IC) FAS-R (this intracellular domain is designated FAS-IC). This new protein is able to modulate the function of FAS-R. In addition, MORT-I is capable of self-association and can activate its own cytotoxicity. The invention also relates to the preparation and use of MORT-I.

It should be noted that the names HF-I (the original designation) and MORT-I (the designation currently used) are both used everywhere and designate the same protein.

BACKGROUND OF THE INVENTION AND BACKGROUND

Tumor necrosis factor (TNF-α and Lymphotoxin (TNF-β) (hereinafter TNF-α and TNF-β will both be referred to as TNF) are multifunctional cytokines that provoke inflammatory processes, synthesized mainly by mononuclear phagocytes, and have a variety of effects on cells (Wallach , D. (1986) in: Interferon 7 (Ion Grosser, ed.), Pp. 83-122, Academic Press, London; and Beutler and Cerami (1987)). TNF-α and TNF-β exert their effects by binding with cell surface receptors Some of these effects are beneficial for the body: for example, they can kill tumor cells or cells infected can be used as an anti-tumor agent, and enhance the antibacterial activity of granulocytes. In this way, TNF is involved in the body's defense against tumors and infectious agents and in the healing of wounds. Thus, TNF can be used as an antitumor agent. In this case, it binds to receptors on the surface of tumor cells and thus initiates the processes leading to the death of a tumor cell. TNF can also be used as an anti-infective.

However, both TNF-α and TNF-β also have harmful effects. There is evidence that excessive production of TNF-α may play a major pathogenic role in a number of diseases. So, it is now known that the effect of TNF-α primarily on the vascular system is the main cause of symptoms of septic shock (Tracey et al., 1986). In some diseases, TNF can cause excessive weight loss (cachexia), inhibiting adipocyte activity and causing anorexia, and therefore it has been called kakhetin. It has also been described as a mediator of tissue damage in rheumatic diseases (Beutler and Cerami, 1987) and as a major mediator of the observed transplant reaction disorder against the host (Piquet et al., 1987). In addition, it is known that TNF is involved in inflammation and in many other diseases.

The initiation and / or modulation of the above biological effects of TNF is carried out by two different, independently expressed, p55 and p75 TNF-R receptors that specifically bind to both TNF-α and TNF-β. These two receptors have structurally different intracellular domains, which indicates the difference in the signals coming from them (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 ai., 1990). However, it is still necessary to elucidate cellular mechanisms, for example, various proteins and possible other factors that are involved in the transmission of intracellular signals from p55 and p75 TNF-R (in PCT / US95 / 05854 and the material presented below, new proteins capable of binding to p75IC and p55IC). It is the intracellular signals that usually occur after binding of the ligand, i.e., TNF (α or β), to the receptor, that are responsible for initiating a cascade of reactions that ultimately lead to the observed cell response to TNF.

Regarding the cytotoxic effect of TNF, in most of the cells currently studied, this action is triggered mainly by the p55 TNF-R receptor. Antibodies against the extracellular domain (ligand binding domain) of p55 TNF-R themselves can trigger a cytotoxic effect (see EP 412486), which correlates with the efficiency of antibody receptor binding. It is believed that this is the first stage in the generation of processes for the production of intracellular signals. A study of mutations (Brakebusch et al., 1992; Tartaglia et al., 1993) showed that the biological function of p55 TNF-R depends on the integrity of its intracellular domain, and accordingly it was suggested that the initiation of an intracellular signal leading to the cytotoxic effect of TNF , is carried out as a result of the connection of two or more intracellular p55 domains of TNF-R. In addition, TNF (α and β) is detected as a homotrimer and, as suggested, induces an intracellular signal through p55 TNF-R due to its ability to bind and form cross-links between receptor molecules, i.e. implement receptor aggregation. PCT / US95 / 05854, as well as the present work, describes how p55IC and p55DD can self-associate and induce ligand-independent effects on the cell that TNF exerts.

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 expressed in various tissues and showing homology to a number of cell surface receptors, including TNF-R and NGF-R. FAS-R mediates cell death by apoptosis (Itoh et al., 1991) and, apparently, serves as a factor in the negative selection of autoreactive T cells, i.e. during T-cell maturation, FAS-R mediates apoptosis of T-cells that recognize their own antigens. Mutations in the FAS-R gene (lpr) have also been found to cause lymphoproliferative disorder in mice, which corresponds to a human autoimmune disease, systemic lupus erythematosus (Watanabe-Fukunaga et al., 1992). The FAS-R ligand, apparently, is a cell surface molecule, which along with other cells are killer T cells (or cytotoxic T lymphocytes - CTL). Therefore, when such CTLs come into contact with FAS-R-bearing cells, they are able to induce apoptosis of FAS-R-bearing cells. Further, monoclonal antibodies against FAS-R were obtained and these monoclonal antibodies were able to induce apoptosis of cells carrying FAS-R, including mouse cells transformed with cDNA encoding human FAS-R (Itoh et al., 1991).

It was also found that various other normal cells along with T-lymphocytes express FAS-R on the surface and can be killed by triggering this receptor. It is believed that the uncontrolled induction of such a process of cell death contributes to tissue damage in certain diseases, for example, destruction of liver cells in acute hepatitis. Therefore, the search for ways to limit the cytotoxic activity of FAS-R may be of therapeutic value.

On the contrary, since it has been found that certain malignant cells and HIV-infected cells carry FAS-R surfaces, anti-FAS-R antibodies or the FAS-R ligand can be used to trigger FAS-R receptor-mediated cytotoxic effects in these cells and thus serve as a means of combating such malignant cells or cells infected with the HIV virus (see Itoh et al., 1991). Therefore, the search for other ways to increase the cytotoxic activity of FAS-R can also be of therapeutic value.

For a long time, it was considered necessary to find a way to modulate the cell response to TNF (α or β) and the FAS ligand, for example, in the pathological cases mentioned above, where there is an increased expression of TNF and the FAS ligand, it is desirable to suppress the cytotoxic effects induced by TNF or the ligand FAS-R. In other cases, for example when healing wounds, it is desirable to increase the effect of TNF, or in the case of FAS-R, it is desirable to increase the effects mediated by FAS-R in tumor cells or cells infected with the HIV virus.

The authors of this invention have used a number of approaches (see, for example, European Applications EP 186833, EP 308378, EP 398327 and EP 412486) to regulate the harmful effects of TNF by inhibiting the binding of TNF to its receptors with anti-TNF antibodies or using soluble TNF receptors (according to essentially essentially soluble intracellular receptor domains) competing with TNF-R receptors on the cell surface for TNF binding. Further, given that binding of TNF to receptors is necessary to induce the effects of TNF on the cell, the inventors have adopted an approach to modulate the effects of TNF based on modulation of TNF-Rs activity. Briefly, EP 568925 (IL 101769) relates to a method for modulating signal transfer and / or cleavage within TNF-Rs, whereby peptides or other molecules can interact with the receptor itself or with the effector proteins involved in the interaction with the receptor, thereby modulating the normal functioning of TNF -R. EP 568925 describes the construction and properties of various p55 TNF-R mutants having mutations in the extracellular, transmembrane and intracellular domains of p55 TNF-R. In this way, regions that are essential for the functioning of the receptor, i.e. for ligand binding (TNF) and subsequent signal transduction and intracellular signal transduction, which ultimately leads to the observed effect of TNF on the cell. Further, a number of approaches are also described for the isolation and identification of proteins, peptides or other factors that are capable of binding to various regions of the above TNF-R domains. These proteins, peptides and other factors may be involved in the regulation or modulation of TNF-R activity. EP 568925 has also set forth a number of approaches for the isolation and cloning of DNA sequences encoding such proteins and peptides; constructing expression vectors for the synthesis of these proteins and peptides; obtaining antibodies or fragments thereof that interact with TNF-R or with the above proteins and peptides that bind different regions of TNF-R. However, EP 568925 does not indicate actually existing proteins and peptides that bind to the intracellular domains of TNF-R (e.g., p55 TNF-R), does not describe an approach using a yeast digibrid system to isolate and identify such proteins and peptides that bind to intracellular domains TNF-R. No proteins or peptides capable of binding the intracellular FAS-R domains have also been previously detected.

Thus, when one wants to suppress the effect of TNF or FAS-R ligand, it is desirable to reduce the amount of TNF-R or FAS-P on the cell surface or their activity, while when trying to increase the effect of TNF or FAS-P ligand, it is desirable to increase the amount of TNF-α R or FAS-R or their activity. For this purpose, the nucleotide sequence of p55 TNF-R and p75 TNF-R promoters was determined and analyzed, and a number of key sequences were found that are characteristic of various transcriptional regulation factors, including those that can control TNF-R expression at the promoter level, those. suppressing reading from a promoter to reduce the number of receptors; and enhancing reading from a promoter to increase the number of receptors (see IL 104355 and IL 109633). Relevant studies related to FAS-R control at the level of the promoter of the FAS-R gene have not yet been published.

Further, the following should be mentioned. It is known that tumor necrosis factor (TNF) receptors and structurally related FAS-R receptors trigger in cells upon stimulation by their ligands synthesized by leukocytes, destructive activities that lead to the death of these cells, but the mechanisms of this launch are still not well understood. Mutation studies show that different regions of their intracellular domains are involved in the generation of cytotoxicity signals by the FAS-R and p55 TNF receptors (p55-R) (Brakebusch et al., 1992; Tartaglia et al., 1993; Itoh and Nagata, 1993). These areas ("death domains") have similar sequences. The death domains of both FAS-R and p55-R tend to self-associate. Their self-association appears to stimulate receptor aggregation, which is necessary for signal initiation (see PCT / US95 / 05854, as well as Song et al., 1994; Wallach et al., 1994; Boldin et al., 1995), and a high level of receptor expression can lead to triggering of signals independent of the ligand (PCT / US95 / 05854 and Boldin et al., 1995).

Thus, prior to the advent of PCT / US95 / 05854 and the present invention, no proteins were known that can regulate the effect of ligands belonging to the TNF / NGF superfamily, such action as TNF or FAS-R ligand exerts on the cell, mediating the processes of generation of intracellular signals . These processes are apparently controlled largely by the intracellular domains (ICs) of receptors from the TNF / NGF receptor superfamily, e.g., TNF-Rs, i.e. intracellular domains p55 and p75 TNF-R (p551C and p751C, respectively), as well as FAS-IC.

In accordance with this, the aim of the present invention is to obtain proteins, namely MORT-I, its analogues, fragments and derivatives, which are able to bind to the intracellular domain of FAS-R. It is believed that these proteins are involved in the transmission of intracellular signals initiated by the binding of the FAS ligand to its receptor. The MORT-I protein, its analogues, fragments and derivatives of the present invention are different from the FAS-IC binding proteins described in the previously mentioned applications.

Another object of the invention is to provide antagonists (e.g. antibodies) against these FAS-IC binding molecules, i.e. against the protein MORT-I, its analogues, fragments and derivatives. These antagonists can be used to suppress signal transduction processes when such FAS-IC binding proteins are positive signal effectors (i.e., induce signals), or to enhance signal transduction when such FAS-IC binding proteins are negative effectors (i.e., suppress signals).

Another objective of the invention is the use of the MORT-I protein, its analogues, fragments and derivatives for the isolation and characterization of other proteins or factors that may, for example, participate in the final links of the signaling process, and / or for the isolation and identification of other receptors, involved in the initial links of the signaling process. These proteins, factors and receptors (for example, other FAS-R or related receptors) could bind to the MORT-I protein, its analogs, fragments and derivatives, and thus participate in their functioning.

In addition, the purpose of this invention is the use of the above protein MORT-I, its analogues, fragments and derivatives as antigens in the preparation of polyclonal and / or monoclonal antibodies against them. These antibodies, in turn, can be used, for example, to purify a new MORT-I protein from other sources, for example, from cell extracts or transformed cell lines.

Further, these antibodies can be used for diagnostic purposes, for example, to identify disorders associated with the abnormal functioning of cellular effects mediated by the FAS-R receptor.

Another objective of the invention is to provide pharmaceutical formulations comprising the MORT-I protein, its analogs, fragments and derivatives, as well as pharmaceutical formulations comprising the above antibodies or other antagonists.

SUMMARY OF THE INVENTION

According to this invention, we have discovered a new protein that is able to bind to the intracellular domain of FAS-R. This FAS-IC binding protein can act as a mediator or modulator of the action of the FAS-R ligand on cells, mediating or modulating the signaling process that usually occurs after binding of the FAS-R ligand to the cell wall.

This new protein is designated as HFI or MORT-I ('Mediator of Receptor Toxicity') and, in addition to its ability to specifically bind to FAS-IC, has other properties (see Example 1), for example, it has a region homologous to the regions of the "death domain" (DD) of p55-TNF-R and FAS-R receptors (p55-DD and FAS-DD) and therefore capable of self-association. MORT-I is also able to activate its own cytotoxicity in the cell, an activity that is possibly related to its ability to self-associate. It has now also been found that co-expression of that region in MORT-I (HFI) that contains a sequence homologous to the “death domain” (MORT-DD present in the C-terminal portion of MORT-I). This region strongly inhibits cell death induced by FAS, as might be expected based on its ability to bind to the “death domain” of FAS-IC. Further, under the same experimental conditions, it was found that the co-expression of the MORT-I region that does not contain the MORT-IDD region (N-terminal portion of MORT-I, amino acids 1 to 117, “head portion of MORT-I”), it does not suppress cell death induced by FAS, and does not increase the cell cytotoxicity induced by FAS to any degree.

Accordingly, the present invention provides a DNA sequence encoding a protein, which is designated here as MORT-I, its analogues or fragments, each of which is able to bind and interact with the intracellular domain of the FAS receptor ligand (FAS-IC).

In particular, this invention provides a DNA sequence from the group including:

(a) a cDNA sequence from a region encoding a MORT-I protein;

(b) a DNA sequence capable of hybridizing with the cDNA of paragraph (a) under moderate conditions. This sequence encodes a biologically active protein that binds the intracellular domain of FAS-R; and

(c) a DNA sequence that is obtained from DNA (a) and (b) as a result of the degeneracy of the genetic code. This sequence encodes a biologically active protein that binds the intracellular domain of FAS-R.

A typical example of the above sequence is a DNA sequence encoding a MORT-I protein, which contains the sequence depicted in Figure 4.

The invention also provides the MORT-I protein, its analogs, fragments, or derivatives encoded by the above sequences of this invention, said proteins, its analogs, fragments, and derivatives being able to bind or interact with the intracellular domain of FAS-R.

A representative example of the above protein of this invention is the MORT-I protein having the amino acid sequence derived from the nucleotide sequence shown in FIG. 4.

The invention also provides vectors encoding the MORT-I protein, its analogues, fragments or derivatives of this invention, which contain the above DNA sequences of this invention, these vectors being able to be expressed in corresponding eukaryotic or prokaryotic host cells; transformed eukaryotic or prokaryotic host cells containing such winds; and a method for producing protein MORT-I, its analogues; appropriate conditions for the expression of these proteins, its analogues, fragments or derivatives; post-transcriptional modification of the aforementioned protein necessary to obtain said proteins, and isolation of the aforementioned expressed proteins, its analogues, fragments or derivatives from the culture medium from the aforementioned transformed cells or from cell extracts of the aforementioned transformed cells.

In another section of the present invention, antibodies or active derivatives or fragments of these antibodies against the MORT-I protein, its analogs, fragments and derivatives of the present invention are also provided.

In yet another section of the invention, various examples of the use of the above DNA sequences or the proteins encoded by them according to this invention are given. Among them are the following examples:

(i) a method for modulating the action of a FAS-R ligand on cells carrying FAS-R, comprising treating the aforementioned cells with the MORT-I protein, its analogs, fragments or derivatives, or combinations thereof according to this invention, each of which is capable of binding to the intracellular domain and modulate the activity of FAS-R. In this method, the processing of cells includes the introduction into the indicated cell of the MORT-I protein, its analogues, fragments or derivatives, or combinations thereof in a form suitable for intracellular administration, or the introduction into the above cell of DNA sequences encoding the above proteins, analogs, fragments or derivatives , or combinations thereof in the composition of the corresponding expression vector carrying the above sequences, while the above vector is able to efficiently integrate into the DNA sequences of the above cells m such that said sequence is expressed in said cell;

(ii) a method for modulating the action of the FAS-R ligand on cells, comprising treating said cells with the MORT-I protein, its analogues, fragments, or derivatives, each of which is capable of binding to the intracellular domain and modifying the activity of FAS-R. The aforementioned cell treatment includes the introduction into the specified cell of the specified protein MORT-I, its analogues, fragments or derivatives in a form suitable for their introduction into the cell, or the introduction of the indicated cell the DNA sequence encoding the specified protein MORT-I, its analogues, fragments or derivatives in the composition of the corresponding vector carrying the specified sequence, while the specified vector is able to effectively integrate the specified sequence in the specified cell so that the specified sequence expressed in the specified cell;

(iii) a method such as in (ii), wherein said treatment of said cells consists of transfecting said cells with a recombinant animal virus vector, comprising the steps of:

(a) constructing a recombinant animal virus vector carrying a sequence encoding a virus envelope protein (ligand) that is capable of binding to a specific receptor on the surface of a cell carrying FAS-R, and a second sequence encoding a protein from the number of MORT-I proteins, its analogues , fragments and derivatives of this invention which, when expressed in said cell, are capable of modulating the activity of said FAS-R; and

(b) infection of said cell with said vector (a);

and or derivatives. When the MORT-I protein or part of it is on the surface of the cell, the composition is prepared in a form for use outside the cell, and when the protein is intracellular, the composition is prepared in a form for administration to the cell;

(v) a method for modulating the effect of a ligand on cells carrying FAS-R, comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence of at least a portion of the MORT-I sequence of this invention, said oligonucleotide sequence being capable of blocking MORT-I;

(vi) a method, such as in (v), where the processing of cells consists in transfecting said cells with a recombinant animal virus vector, comprising the following steps:

(a) constructing a recombinant animal virus vector carrying a sequence encoding a virus envelope protein (ligand) that is capable of binding to a specific receptor on the surface of a cell carrying FAS-R, and a second sequence that is an oligonucleotide sequence encoding an antisense sequence of at least part of the MORT-I sequence according to the invention, wherein said oligonucleotide sequence is capable of blocking the expression of the MORT-I protein during iv eating said virus in said cell; and

(b) infection of said cell with said vector (a);

(vii) a method for treating tumor cells or cells infected with HIV or other desired cells, comprising:

(a) constructing a recombinant animal virus vector carrying a sequence encoding a virus envelope protein that is capable of binding to a receptor on the surface of a tumor cell, or to a receptor on the surface of a cell infected with HIV virus, or to a receptor carried by other desired cells, and a second a sequence encoding one of the MORT-I proteins, its analogues, fragments and derivatives of this invention, which, when expressed in a specified tumor cell, a cell infected with HIV virus, or some other an empty cell is capable of killing the specified cell; and

(b) infection of said tumor cell; HIV virus infected cells; or some other cell specified vector (a);

and which provides expression of the indicated ribozyme sequence in the specified cell. In this method, when expressing the indicated ribozyme sequence, it interacts with the indicated cell mRNA sequence and cleaves the indicated mRNA sequence, leading to the suppression of the expression of the MORT-I protein in these cells;

(ix) a method of the above, wherein said MORT-I protein or a sequence encoding said MORT-I protein includes at least that portion of the MORT-I protein that specifically binds to FAS-IC, or at least that portion a sequence that encodes a portion of a MORT-I protein specifically binding to FAS-IC;

(x) a method for isolating and identifying a protein capable of binding to the intracellular domain of FAS-R, comprising applying a non-stringent Southern hybridization procedure followed by PCR cloning in which the sequence or parts thereof according to this invention are used as probes to bind to cDNA regions or portions of DNA from a gene library that have at least partial homology with these probes. These linked sequences were then amplified and cloned using the PCR procedure. The result was clones encoding a protein having at least partial homology with the indicated sequences according to this invention.

The present invention also provides a pharmaceutical composition for modulating the action of a FAS ligand on a cell, comprising one of the following additives as an active ingredient:

(i) the MORT-I protein of this invention, its biologically active fragments, analogues, derivatives or mixtures thereof; (ii) a recombinant animal virus vector encoding a protein capable of binding to a cell surface receptor and encoding a MORT-I protein or biologically active fragments or analogues thereof according to this invention; and (iii) an oligonucleotide sequence encoding the antisense sequence of the MORT-I sequence of this invention, wherein said oligonucleotide sequence may be a second sequence of the above (ii) recombinant animal virus vector.

It should be emphasized that MORT-I has a special region that binds to FAS-IC, and another separate region that is involved in the self-association of MORT-I, and, accordingly, individual regions or parts of MORT-I can be independently used for identifying other proteins, receptors, and the like that are capable of binding to MORT-I or FAS-R and may be involved in the transmission of intracellular signals due to MORT-I or FAS-R. Further, MORT-I may have other types of activity associated with either the above or other areas of MORT-I, or combinations thereof, for example, enzymatic activity related to the effects of MORT-I on the cell’s own cytotoxicity. Thus, MORT-I can also be used for the specific identification of other proteins, peptides, etc., which may be involved in the manifestation of such additional activities in MORT-I.

The following detailed description of the present invention also gives other aspects and applications thereof.

It should be noted that the terms “Modulation of the action of the FAS ligand on the cell” and “Modulation of the action of the MORT-I on the cell” as used herein mean processing both in vitro and in vivo.

Brief Description of the Drawings

Figure 1 A and B reproduce the autoradiogram of SDS-PAGE gels (10% acrylamide), showing the interaction between HFI (MORT-I) and FAS-IC in vitro. In FIG. 1A shows a control autoradiogram of protein immunoprecipitate (from HeLa cell extracts transfected with a FLAG-NFI fusion protein (FLAG-MORT-I) or luciferase cDNA (control), wherein immunoprecipitation was performed using anti-FLAG antibodies; and

Figure 1B shows a characteristic gel autoradiogram reflecting the interaction between HFI and FAS-IC in vitro using autoradiographic analysis of the binding of metabolically labeled / ³⁵ S / -methionine HFI synthesized in transfected HeLa cells as a protein fused to the FLAG octapeptide ( FLAG-MORT-I), with GST, human and mouse GST-FAS-IC fusion protein (GST-huFAS-IC, GST-mFAS-IC) and GST-FAS-IC fusion proteins in which FAS-IC contains a mutation substitution Ile on Ala at position 225 (GST-mFAS-IC 1225A). Proteins from HeLa cells labeled at / ³⁵ S /, including the labeled FLAG-MORT-I fusion protein, were first extracted and then allowed to interact with various GST and GST-FAS-IC proteins (attached to glutathione beads) and then separated with using SDS-PAGE. In all control experiments, extracts from HeLa cells transfected with luciferase interacted with GST and GST-FAS-IC fusion proteins and performed SDS-PAGE. FIG. 1A and 1B are also described in Example 1.

2A, B and C reproduce autoradiograms of SDS-PAGE gels (10% acrylamide), in which various immunoprecipitates from transfected HeLa cells were separated. They show the in vivo interaction of HF-I with FAS-IC. HeLa cells were transfected with DNA constructs encoding: only HFI-FLAG fusion protein (FLAG-MORT-I), human HFI-FLAG fusion protein and FAS-R (FLAG-MORTI + FAS / APOI) or only human FAS-R (FAS / APOI) (Fig. 2A); or a human HFI-FLAG and p55-R fusion protein (FLAG-MORTI + p55-R) (Fig. 2B); or an HFI-FLAG fusion protein and a chimeric fusion protein from human FAS-R and p55 in which the extracellular domain of FAS-R has been replaced by the corresponding region from p55-R (FLAG-MORTI + chimera p55-FAS), or only a chimeric fusion protein FAS-R-p55-R (Fig. 2C). In all cases, transfected cells were metabolically labeled with / ³⁵ S / -cysteine (20 μCi / ml) and / ³⁵ S / -methionine (40 μCi / ml) and the protein was extracted. Protein extracts from various transfected cells were then immunoprecipitated with various antibodies: anti-FLAG, anti-FAS, anti-p75-R and anti-p55-R (FLAG, αFAS, αp75-R and αp55-R in FIG. 2A-C) and shared SDS-PAGE. 2A, protein bands corresponding to FAS-R (Fas / APOI) and HFI-FLAG (FLAG-MORTI) are visible on the left; between FIGS. 2A and B, the relative positions of standard molecular weight (kD) markets are shown; and in FIG. 2C, protein bands corresponding to p55R and p55-FAS chimera are visible on the right. 2A-C are also described in Example 1.

Figure 3 reproduces Northern blotting in which poly A + RNA (0.3 μg) from transfected HeLa cells was detected using HF-I cDNA probes as described in Example 1.

Figure 4 gives a schematic representation of a preliminary nucleotide sequence and the HF-I amino acid sequence obtained from it, as described in Example 1, in which the "death domain" is underlined as a possible translation start site, i.e. the methionine residue at position 49 is underlined (M, underlined by a bold line). An asterisk indicates the translation stop codon (nucleotides 769-771). At the beginning and middle of each line are two numbers indicating the relative positions of nucleotides and amino acids in this sequence, counted from the beginning of the sequence (5′-end), where the first number indicates the position of the nucleotide and the second the amino acids.

Figure 5 presents the results of experiments to determine the C-terminus of MORT-I, where Figure 5 reproduces a gel autograph of the gel after SDS-PAGE (10% acrylamide), which was used to separate the various MORTI-FLAG fusion proteins expressed in HeLa cells and metabolically labeled with ³⁵ S-cysteine and ³⁵ S-methionine followed by immunoprecipitation or with anti-FLAG monoclonal antibodies (M2) (lanes 2, 4 and 6) or, in control, with antibody-p75 TNF-R (# 9 ) (lanes 1, 3 and 5) as described in Example 1.

6 (A and B) graphically depicts ligand-independent triggering of cytotoxic effects in cells transfected with MORT-I, where cell viability was determined either in a neutral red absorption test (FIG. 6A), or in the case of a specific determination of cell viability, expressing transfecting DNA - by measuring the amount of placenta secreted into the medium of alkaline phosphatase (Fig. 6B). HeLa cells were transfected with vectors encoding HFI (MORTI), human FAS-IC, human p55-IC, or luciferase (control). The expression of these vectors is controlled by tetracycline. In all cases, these cells were also transfected with a cDNA preparation encoding secreted alkaline phosphatase, which allows one to detect the effect of transient expression of these proteins on cell viability. 6A and 6B, light bars represent transfected cells growing in the presence of tetracycline (1 μg / ml, for expression block), and filled bars represent transfected cells growing in the absence of tetracycline. 6A and B are described in Example 1.

FIG. 7 provides a graphical representation of the effect of various portions of the MORT-I protein on the cytotoxic effects of a cell mediated by the FAS-R receptor, as described in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a new protein MORT-I (HFI), which is able to bind to the intracellular domain of the FAS-R receptor belonging to the TNF / NGF superfamily, and therefore is considered as a mediator or modulator of the FAS-R receptor, which functions, for example, in the signaling process initiated by binding of the FAS ligand to FAS-R. The amino acid sequences and DNA sequences for MORT-I according to this invention are new sequences; they are not contained in the databank for DNA or amino acid sequences in "GENEBANK" or "PROTEIN BANK".

Thus, the present invention describes a DNA sequence encoding a MORT-I protein and a MORT-I protein encoded by this DNA sequence.

In addition, the present invention describes DNA sequences encoding biologically active analogues, fragments and derivatives of the MORT-I protein, and analogues, fragments and derivatives encoded by these DNA sequences. The preparation of such analogues, fragments, and derivatives is carried out using a standard procedure (see, for example, Sambrook et al., 1989), which allows one to divide, insert, or replace one or more codons in the DNA sequence encoding the MORT-I protein, which results to obtain analogues having, in comparison with a natural protein, at least one altered amino acid. Also suitable are analogs that retain at least the ability to bind to the intracellular domain of FAS-R, or analogues that can mediate either any other binding or enzymatic activity, for example, analogues that bind FAS-IC but do not give signals, i.e. e. do not bind to a receptor, protein, or other factor in the next link in the reaction, or do not catalyze a signal-dependent reaction. In this way, you can get an analog that has a so-called dominant negative effect, namely an analog that has a defect either in binding to FAS-IC or in subsequent signal transmission after binding. Such analogs can be used, for example, to suppress the action of the FAS ligand by competition with natural FAS-IC binding proteins. In the same way, one can obtain the so-called dominant-positive analogues, which can serve to enhance the effect of the FAS ligand. They may have the same or better FAS-IC binding characteristics and the same or better signaling characteristics than natural FAS-IC binding proteins. In a manner similar to that described for the preparation of MORT-I analogues, biologically active fragments of MORT-I can be obtained. Acceptable are those fragments of MORT-I that retain the ability to bind FAS-IC, or those that mediate either binding or enzymatic activity, as noted above. In accordance with this can be obtained fragments of MORT-I, which have a dominant negative or dominant positive effect, as was noted in relation to analogues. It should be noted that these fragments represent a special class of analogues of this invention, namely, they are defined as MORT-I regions taken from the complete MORT-I sequence, each such region or fragment having the above desirable activities. In the same way, derivatives can be obtained using standard modifications of the side groups in one or more amino acid residues of the MORT-I protein, its analogues or fragments, or by conjugation of the MORT-I protein, its analogues or fragments with another molecule, for example, an antibody, enzyme, receptor, etc., as is known to specialists.

The new MORT-I protein, its analogues, fragments and derivatives have a number of possible uses.

(i) They can be used to mimic or enhance the function of the FAS-R ligand in situations where it is desirable to enhance the action of the FAS-R ligand, for example, when using antitumor, anti-inflammatory or antiviral (anti-HIV) antibodies, when it is desired to obtain ligand-induced cytotoxicity FAS-R. In this case, the MORT-I protein, its analogues, fragments or derivatives that increase the effect of the FAS-R ligand, i.e. The cytotoxic effect can be introduced into the cell using standard procedures known as perse. For example, since the MORT-I protein is intracellular and should only be introduced into cells when they want to get the FAS-R ligand effect, a system is needed to specifically introduce this protein into these cells. One way to do this is to construct a recombinant animal virus, such as that obtained from vaccinia, with DNA that contains the following two genes: a gene encoding a ligand that binds to cell surface proteins expressed by cells, such as the AID virus gp120 protein ( HIV), which specifically binds to certain cells (CD 4 lymphocytes and related leukemias), or some other ligand that specifically binds to cells carrying FAS-R, so that the recombinant viral vector has l the ability to bind to cells bearing FAS-R; and a gene encoding a MORT-I protein. Thus, when expressing the envelope protein of a virus that binds to the cell surface, a tumor cell or other cell carrying FAS-R will become a target for this virus, as a result of this, the MORT-1 protein will be introduced into these cells using the virus and expression in these cells, it will lead to an increase in the action of the FAS-R ligand, leading to the death of a tumor cell or other cell carrying the FAS-R that they want to kill. Such a recombinant animal virus is constructed using a standard procedure (see, for example, Sambrook et al., 1989). Another possibility is to introduce the MORT-1 protein sequence in the form of oligonucleotides that can be absorbed and expressed by the cell. Another possibility is to modify the method described by Lin et al. In Journal of Biological Chemistry, Vol. 270, No.24, pp. 14255-14258, 1995.

(ii) They can be used to suppress the action of the FAS-R ligand, for example, in case of tissue damage during septic shock, graft rejection reaction against the host, or in acute hepatitis, when it is desirable to block the intracellular signal induced by binding of the FAS-R ligand with FAS-R. In this case, for example, oligonucleotides having the antisense coding sequence of the MORT-1 protein can be introduced into cells using a standard procedure. It will effectively block the translation of mRNA encoding the MORT-1 protein and, thus, block its expression and lead to inhibition of the action of the FAS-R ligand. Such oligonucleotides can be introduced into cells using the above approach using a recombinant virus, in which the second sequence included in this virus is an oligonucleotide sequence.

Another possibility is the use of antibodies against the MORT-1 protein, which inhibit its activity during the transmission of intracellular signals.

Another recently developed method for suppressing the action of the FAS ligand is based on the use of ribozyme. Ribozymes are catalytically active RNA molecules that specifically cleave RNA. You can create ribozymes that cleave a specific RNA, for example, mRNA encoding the MORT-1 protein of the present invention. Such ribozymes must have sequences specific for MORT-1 mRNA and have the ability to interact with it according to a complementary mechanism with subsequent cleavage of this mRNA. This leads to a decrease (or complete loss) of expression of the MORT-1 protein, and the level of decrease in expression depends on the level of expression of the ribozyme in the target cells. Any suitable vector, for example, a plasmid, animal virus vectors (retro viruses), which are usually used for this purpose, can be used to introduce ribozymes into desired cells (e.g., carrying FAS-R) (see above (i), where the virus as the second cDNA sequence encoding a ribozyme with the selected sequence). (For reviews, methods, etc. regarding ribozymes, see Chen et al., 1992; Zhao and Pick, 1993; Shore et al., 1993; Joseph and Burke, 1993; Shimayama et al., 1993; Cantor et al., 1993; Barinaga, 1993; Crisell et al., 1993 and Koizumi et al., 1993).

(iii) They can be used to isolate, identify, and clone other proteins that are capable of binding to them, for example, other proteins involved in the chain of intracellular signal transduction processes induced by the intracellular domain of TNF-R or FAS-R. For example, the MORT-1 protein, namely the DNA sequence encoding it, can be used in the yeast digibrid system (see below. Example 1), in which the sequence for the MORT-1 protein will be used as a “bait” for isolation, cloning and identification in cDNA or DNA of a gene library of other sequences (“victims”) encoding proteins that can bind to MORT-1. In the same way, it can be determined whether the MORT-1 protein of the present invention can bind to other cellular proteins, for example, other receptors from the TNF / NGF receptor superfamilies.

(iv) The MORT-1 protein, its analogs, fragments, or derivatives can also be used to isolate, identify, and clone other proteins of the same class, i.e. those that bind to the intracellular domain of FAS-R or to functionally related receptors and are involved in the transmission of intracellular signals. For this purpose, the aforementioned yeast digibrid system can be used or a recently developed system based on the use of weak Southern hybridization followed by PCR cloning (Wilks et al., 1989). A publication by Wilkes et al. Describes the identification and cloning of putative protein tyrosine kinases using non-strict Southern hybridization followed by PCR cloning based on the known kinase-specific sequence of the main part of the kinase. According to the present invention, this approach can be applied using a sequence encoding a MORT-1 protein to isolate and clone related proteins that bind the intracellular FAS-R domains.

(v) Another approach using the MORT-1 protein, its analogues, fragments, or derivatives is to use them in affinity chromatography methods to isolate and identify other proteins or factors with which they are able to bind, for example, other receptors related to the FAS-β receptor R, or other proteins or factors involved in the transmission of intracellular signals. With this use of the MORT-1 protein, its analogues, fragments, or derivatives of the present invention, each of them individually can be attached to affinity chromatography matrices. Then, extracts, or isolated proteins, or factors presumably involved in the transmission of intracellular signals are incubated with these immobilized proteins. After completion of the affinity chromatography procedure, these other proteins and factors that have associated with the MORT-1 protein, its analogs, fragments, or derivatives of the present invention can be eluted, isolated, and characterized.

(vi) As noted above, the MORT-1 protein, its analogues, fragments, or derivatives of this invention can also be used as immunogens (antigens) to produce antibodies against them. These antibodies can also be used for purification of the MORT-1 protein either from cell extracts or from lines of transformed cells synthesizing the MORT-1 protein, its analogues or fragments. Further, these antibodies can be used for diagnostic purposes in identifying disorders associated with abnormal functioning of the FAS ligand system, for example, with cellular effects induced by the overactive or low activity FAS-R ligand. Thus, in disorders associated with the abnormal functioning of the intracellular signaling system in which the MORT-1 protein is involved, such antibodies will serve as an important diagnostic tool.

(vii) MORT-1 can also be used as an indirect modulator of a number of other proteins due to its ability to bind to other intracellular proteins (the so-called MORT-1 binding proteins), which in turn directly bind other intracellular proteins or the intracellular domain of a transmembrane protein. An example of such a protein or such an intracellular domain is the well-known TNF p55 receptor, the intracellular signals from which are modulated by a number of other proteins that directly bind to its intracellular domain (see application IL 109632). In fact, we isolated such a protein that binds MORT-1 (see below and Example 2), which binds to the intracellular domain of the TNF p55 receptor.

In order to modulate these other intracellular proteins or intracellular domains of transmembrane proteins, the MORT-1 protein can be introduced into the cell using a number of methods mentioned above (ii).

It should also be noted that the isolation, identification and characterization of the MORT-1 protein of this invention can be performed using various well-known screening procedures. For example, one of these screening procedures, the yeast digibrid procedure set forth herein (Example 1), was used to identify the MORT-1 protein of this invention. In the same way as described above and further, other procedures can be used, for example affinity chromatography, DNA hybridization procedures and other known procedures, for isolating, identifying and characterizing the MORT-1 protein of this invention or for isolating, identifying and characterizing other proteins, factors, receptors, and the like, which are capable of binding to the MORT-1 protein of this invention.

Further, it should also be emphasized that the properties of the MORT-1 protein include its ability to bind to FAS-IC, as well as its ability to self-associate. MORT-1 is also able to activate its own cytotoxicity of the cell, an activity associated with its ability to self-associate. Apparently (see Example 1), the MORT-1 site that binds to FAS-IC is different from the MORT-1 site that is involved in self-association. MORT-1 may also have other types of activity associated with the function of the various aforementioned portions of the MORT-1 molecule, or some other parts of the molecule, or various combinations thereof. These other types of activity can be enzymatic or relate to the binding of other proteins (for example, MORT-1 binding proteins, or other receptors, factors, etc.). Thus, MORT-1 can be used in the above methods of modulating the action of the FAS-R ligand or cellular effects mediated by MORT-1, or it can be used to modulate other cellular signaling processes associated with other receptors, factors, etc. .

More specific in this aspect of the invention is that the DNA molecule itself encoding MORT-1, or its mutant form (i.e., DNA encoding analogues or active fragments of MORT-1) can be used in gene therapy (i.e. by the methods described in (i) and (ii) above to modulate the activity of FAS-R (or modulate or mediate the action of the FAS ligand on cells) In addition, since MORT-1 also has a cytotoxic effect on cells, these DNA molecules or their mutant variants encoding MORT-1 can be used in gene therapy for the module tion MORT-1 actions on cells (including the methods described above in (i) and (ii)) In these applications for gene therapy MORT-1, its analogs and derivatives may be used in three ways.:

(a) the whole MORT-1 protein, its analogues, derivatives or active fragments that are capable of binding to both FAS-IC and MORT-1 (i.e., contain two MORT-1 regions, one of which is involved in binding with FAS-IC, and the other is involved in the self-association of MORT-1), can be used to modulate the effects associated with FAS-R and MORT-1;

(b) a portion of the MORT-1 protein, analogs, derivatives, and active fragments of this portion of MORT-1 that bind to FAS-IC, can be used to induce a “dominant negative” effect on FAS-IC, i.e. to suppress cellular effects mediated by FAS-R, or can be used to induce a “build-up function" of action on FAS-IC, i.e. increased cellular effects mediated by FAS-R; and

(c) a portion of the MORT-1 molecule, analogs, derivatives, and active regions of this portion that specifically bind to MORT-1, can be used to induce "dominant negative" effects on MORT-1, i.e. either suppressing or increasing the cellular effects associated with MORT-1.

As described above in (vi), the MORT-1 protein can be used to produce antibodies to MORT-1. These antibodies or fragments thereof can be used, as described in detail below, it being understood that these uses of the antibodies or fragments thereof relate specifically to MORT-1.

As for the antibodies mentioned here, the term "antibodies" everywhere refers to polyclonal antibodies; monoclonal antibodies (mAb); chimeric antibodies; idiotypes of antibodies (anti-Id) against antibodies that can be labeled in soluble or bound form; as well as their fragments obtained using known methods, for example, such as enzymatic cleavage, peptide synthesis or recombinant technology and others.

Polyclonal antibodies are a heterogeneous population of antibody molecules from animal sera immunized with an antigen. Monoclonal antibodies contain a substantially homogeneous population of antibodies to these antigens, in which all antibodies contain binding sites to essentially the same epitope. MAb can be obtained using methods known to specialists in this field. See, for example, Kohler and Milstein, Nature, 256: 495-497 (1975); US Patent No. 4,376,110; Ausubel et al., Eds., Harlow and Lane ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., eds .. Current Protocols in Immunology, Greene publishing Assoc. and Wiley Interscience N.Y. (1992, 1993), it contains an almost complete list of references on this issue. Such antibodies can belong to any class of immunoglobulins, including IgG, IgM, IgE, IgA, GILD, and any of their subclasses. Hybridomas synthesizing the mAbs of the invention can be cultured in vitro, in situ or in vivo. Currently, in vivo or in situ mAd production methods are preferred due to the high titer of antibodies synthesized using these methods.

Molecules of chimeric antibodies are composed of different regions originating from different animal species, for example, have a variable region of murine mAb and a constant region of human immunoglobulin. Chimeric antibodies are mainly used to reduce the immunogenicity of their use and to increase the yield of their production, for example, when the yield of mouse mAbs from hybridomas is higher, but their immunogenicity is higher in humans, then human / mouse chimeric mAbs are used. Chimeric antibodies and methods for their preparation are known to those skilled in the art (Cabilly et al., Proc. Natl. Acad. Sci. USA 81: 3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851- 6855 (1984); Boulianne et al., Nature 312: 643-646 (1984); Cabilly et al., European Patent Application 125023 (published November 14, 1984); Neuberger et al., Nature 314: 268-270 (1985) ; Taniguchi et al., European Patent Application 171496 (published February 19, 1985); Morrison et al., European Patent Application 173494 (published March 5, 1986); Neuberger et al., PCT Application WO 8601533 (published March 13, 1986); Kudo et al., European Patent Application 184187 (published June 11, 1986); Sahagan et al., J. Immunol. 137: 1066-1074 (1986); Robinson et al., International Patent Application No. WO 8702671 (published May 7, 1987 ); Liu et al., Proc. Natl. Acad. S ci. USA 84: 3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci USA 84: 214-218 (1987); Better et al., Science 240: 1041-1043 (1988); and Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, given above). This list contains almost all references on this issue.

Antibodies against idiotypes (anti-Id) are antibodies that recognize unique determinants typically found in the antigen-binding region of an antibody. Antibodies against Id can be obtained by immunizing animals of the same species and genotype (for example, a line of mice) as an animal serving as a source of mAb with monoclonal antibodies mAb against which anti-Id is prepared. Immunized animals will recognize and respond to the idiotypic determinants of antibodies taken for immunization and synthesize antibodies to these idiotypic determinants (anti-Id antibodies). See, for example, US Patent No. 4699880, which includes almost all the links on this issue.

Anti-Id antibodies can also be used as an “immunogen” to induce an immune response in other animals synthesizing so-called anti-anti-Id antibodies. Anti-anti-Id antibodies can recognize epitopes identical to the epitopes of the parent mAbs that induced anti-Id. Thus, using antibodies to the idiotypic determinants of mAb, other clones expressing antibodies with identical specificity can be identified.

Accordingly, mAbs obtained against the MORT-1 protein, its analogues, fragments or derivatives of the present invention can be used to induce anti-Id antibodies in appropriate animals, for example BALB / c mice. Cells from the spleen of such immunized animals are used to produce hybridomas secreting anti-Id mAbs. Further, anti-Ib mAb can be attached to a carrier, for example, fissurella hemocyanin (KLH) and used to immunize other BALB / c mice. The serum from these animals will contain anti-anti-Id antibodies that have the binding properties of the parent mAbs against the epitope of the aforementioned MORT-1 protein, its analogues, fragments or derivatives.

Thus, anti-Id mAbs have their own idiotypic epitopes, or “idiotopes,” structurally similar to the identified epitopes, for example, as in GRB protein-α.

The term “antibody” refers to both intact molecules and fragments thereof, for example, Fab and F (ab ′) ₂ , which are capable of binding an antigen. Fab and F (ab ′) ₂ fragments lacking the Fc fragment of an intact antibody, for obvious reasons, go out of circulation faster and may have less tissue specificity than intact antibodies (Wahl et al., J. Nucl. Med. 24: 316- 325 (1983)).

It should be understood that Fab and F (ab ′) ₂ fragments and other antibody fragments suitable for the present invention can be used to detect and quantify the MORT-1 protein using the methods proposed here for intact antibody molecules. Such fragments are usually obtained by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ′) ₂ fragments).

An antibody is said to be “capable of binding” to a molecule if it is capable of specifically reacting with the molecule by binding to it. The term "epitope" means a portion of any molecule that can be recognized by this antibody and bind to it. Epitopes or “antigenic determinants” usually consist of chemically active moieties on the surface of a molecule, such as amino acids or sugar side chains, and have a specific three-dimensional structure as well as a specific charge.

An “antigen” is a molecule or part of a molecule that is capable of binding an antibody and, in addition, capable of inducing an animal to synthesize antibodies capable of binding to an epitope of this antigen. An antigen may have one or more epitopes. The above specific reaction means that the antigen will react in a highly selective manner with the corresponding antibody, but will not react with many other antibodies that can be induced by other antigens.

Antibodies, including antibody fragments needed for the present invention, can be used to quantify and qualitatively determine MORT-1 in a sample, or to detect cells that express MORT-1 of the present invention. This can be done using immunofluorescence assay methods using antibodies labeled with fluorescent dyes (see below) using light microscopy, flow cytometry, or fluorometry.

Antibodies (or fragments thereof) suitable for the present invention can be used for histological examination using immunofluorescence or immunoelectronic microscopy, for example for in situ detection of MORT-1 of the present invention. In situ detection can be performed by obtaining a histological sample from a patient and treating this sample with labeled antibodies of the invention. A biological sample is either directly treated with a labeled antibody (or fragment) against a given antigen, or first with a corresponding unlabeled antibody, and then a second labeled antibody against the first antibody. Using this procedure, it is possible to determine not only the presence of MORT-1, but also its distribution in the test tissue. Using this invention, any person skilled in the art will recognize that any variety of histological methods (e.g., staining procedures) can be modified for in situ detection.

Such MORT-1 tests of the present invention typically include incubation of a biological sample, such as biological fluid, tissue extract, freshly isolated cells, such as lymphocytes or white blood cells, or cells incubated in culture in the presence of the desired amount of labeled antibodies capable of identifying the MORT protein -1, and the detection of these antibodies by any of a number of well-known methods.

The biological sample can be applied to a solid support or carrier, for example nitrocellulose or another solid support or carrier, which are capable of immobilizing cells, cell particles or soluble proteins. This substrate or carrier can then be washed with an appropriate buffer and then treated with labeled antibodies of the invention as described above. The rigid support or carrier can then be washed a second time with buffer to remove unbound antibodies. The amount of bound label on said substrate or carrier can then be detected by appropriate methods.

"Solid phase support", "solid phase carrier", "rigid substrate", "hard carrier", "substrate" or "carrier" are used to denote a substrate or carrier capable of binding an antigen or antibody. Well-known substrates or carriers are glass, polystyrene, polypropylene, polyethylene, dextran, amylase nylon, natural or modified cellulose, polyacrylamides, gabro and magnetites. For the purposes of this invention, natural carriers may be somewhat soluble or insoluble. The substrate material may have any possible structural form, but such that its molecules have the ability to bind to an antigen or antibody. So, the shape of the substrate or carrier can be spherical in the form of balls, cylindrical with the inner surface of the calibrated tube or with the outer surface of the stick, or their surface can be flat, like a sheet of calibrated strip, etc. They prefer substrates or carriers in the form of polystyrene balls. Other convenient carriers for binding antibodies or antigens may be known to those skilled in the art, or they may select them by routine experimental testing.

The binding activity of a given batch of antibodies of this invention, as noted above, can be determined using well-known methods. The specialist can determine the operational and optimal conditions for this type of test, using routine experimental checks.

Other steps may be introduced into these tests, such as washing, stirring, shaking, filtering, etc., as is customary or as necessary in each case.

One of the ways in which an antibody of the invention can be labeled is by attaching an enzyme to it and using an enzyme immunoassay (EIA). This enzyme will then react with this substrate when interacting with the appropriate substrate, and the resulting chemical reaction product can be detected, for example, by spectrophotometry, fluorometry or visually. Enzymes that are used to label antibodies include, for example, malate dehydrogenase, staphylococcus nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, glucose permease, alkaline phosphatase, glucose permease, , glucose-6-phosphate dehydrogenase, glucoamylase, acetylpholinesterase and other enzymes.

The detection of antibodies can be performed using chromometric methods based on the detection of a chromogenic substrate of the enzyme. Detection can also be done by visually comparing the magnitude of the enzymatic reaction of the substrate with a standard sample prepared in a similar way.

The detection of antibodies can also be performed using various other methods of immunological analysis. For example, using radiolabeled antibodies or antibody fragments, R-PTPase can be detected by radioimmunoassay (PIA). A good description of PIA can be found in the book. Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al., North Holland Publishing Company, NY (1978), specifically referring to the chapter "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., for relevant references. A radioactive isotope can be recorded using gamma counters, scintillation counters, or using autoradiography.

Antibodies of the present invention can also be labeled with a fluorescent compound. Fluorescently labeled antibodies when exposed to light of the appropriate wavelength can be detected by their fluorescence. The most commonly used fluorescent label compounds are fluorescein, isothiocyanate, rhodamine, phycoerethrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluoresceamine.

Antibodies can also be labeled with fluorescent metals, such as ¹⁵² E or others in the lanthanide series. These metals can be attached to the antibody using metal chelating groups, for example diethylene triamine pentaacetic acid (ETPA).

An antibody can also be labeled by attaching a chemiluminescent compound to it. The presence of an antibody labeled with a chemiluminescent substance can be determined by the presence of fluorescence that occurs during a chemical reaction. Examples of chemiluminescent compounds which are particularly suitable as labels are luminol, isoluminol, acridyl hydrochloride, imidazole, acridinium acid ester, oxalic acid diethyl ester.

The antibody of the invention can also be labeled with a compound capable of bioluminescence. Bioluminescence is a type of chemiluminescence found in biological systems in which a protein catalyst enhances the chemiluminescence reaction. The presence of a bioluminescent protein is determined by the presence of luminescence. Important bioluminescent compounds as labels are luciferin, luciferase and aquarin.

The antibody molecules of the present invention can be adapted for use in an immunometric assay, also known as a "two-site" or "sandwich" test. In a typical immunometric test, a certain amount of unlabeled antibodies (or antibody fragments) is bound to a rigid support or carrier, and then a certain amount of labeled antibodies is added, which allows one to detect and / or determine the amount of the triple complex formed by the solid phase antibody, antigen and labeled antibody.

A typical and preferred immunometric test includes an “early” test, in which the antibody bound to the solid phase is first reacted with the test sample, which allows the antigen to be extracted from the sample by forming a binary antibody-antigen complex in the solid phase. After the required incubation period, the solid support or carrier is washed to remove the liquid sample material, including the unreacted antigen, if any, and immersed in a solution containing an unknown amount of labeled antibodies (which function as “signaling molecules”). After a second incubation period in which labeled antibodies complex with an antigen bound to a solid support or carrier with unlabeled antibodies, the solid support or carrier is washed a second time to remove unreacted labeled antibodies.

In another type of “sandwich” test, which may also be suitable for the antigens of the present invention, so-called “one-time” and “reverse” tests are used. A single test involves one incubation step, as both antibodies bound to a solid support or carrier and labeled antibodies are simultaneously added to the test sample. After the incubation period has ended, a solid support or carrier is washed to remove the remainder of the liquid sample and unreacted labeled antibodies. The presence of labeled antibodies bound to a solid support or carrier is then determined, as is done in the “early” sandwich test.

The reverse test uses the stepwise addition of a solution of labeled antibodies to the liquid sample first, then, after the required incubation period, unlabeled antibodies bound to a solid support or carrier are added. After a second incubation period, the solid phase is washed in an appropriate manner to remove residual test sample and unreacted labeled antibodies. The determination of labeled antibodies bound to a solid support or carrier is then carried out as in the “early” and “one-time” tests.

MORT-1 of this invention can be obtained using a standard recombinant technology procedure (see, for example, Sambrook, et al., 1989), in which the corresponding eukaryotic or prokaryotic host cells are transformed with suitable eukaryotic or prokaryotic vectors containing sequences encoding these proteins . In accordance with this invention also relates to such expression vectors and transformed host cells for the synthesis of proteins of this invention. As mentioned above, these proteins also include their biologically active analogues, fragments and derivatives, and accordingly, the vectors encoding them include vectors encoding analogues and fragments of these proteins, and the transformed host cells include cells synthesizing such analogues and fragments. Derivatives of these proteins are derivatives obtained by standard modification of proteins, their analogues or their fragments synthesized by transformed host cells.

The present invention also relates to pharmaceutical formulations that include recombinant animal virus vectors encoding the MORT-1 protein. These vectors also encode a virus envelope protein capable of specifically binding to surface proteins of a target cell (eg, a tumor cell) so that the sequence for MORT-1 is introduced into the cell. Other aspects of this invention are given in the following examples.

Now this invention will be described in more detail in the following non-limiting examples and the corresponding figures.

EXAMPLE 1: CLONING AND ISOLATION OF THE MORT-1 PROTEIN THAT BINDS THE EXTRACELLULAR DOMAIN FAS-R

(i) Screening in the Hybrid System and Test of Expression of β-Galactosidase in the Hybrid System

To isolate proteins interacting with the intracellular domain of FAS-R, a yeast digibrid system was used (Fields and Song, 1989; see also joint applications IL 109632, 112002 and 112692). Briefly, this digrid system is a genetic analysis in yeast cells that allows you to detect specific in vivo protein-protein interactions to restore the function of a eukaryotic transcription activator, such as GAL4, which has two different domains - a DNA binding domain and an activation domain. When these domains are expressed and reunited, the resulting reduced GAL4 protein is able to bind to the 5th region of the activating sequence, which in turn activates a promoter that controls the expression of the signal gene, for example, lac Z or HIS3. The expression of these genes is easily observed in cultured cells. In this system, the genes of two proteins, the interaction of which is studied, are cloned in two different expression vectors. In one expression vector, the sequence for one of these proteins is cloned together with the sequence for the GAL4 DNA binding domain, resulting in a fusion protein containing the GAL4 DNA binding domain. In another vector, the sequence for another protein is cloned together with the GAL4 activation domain. These two vectors together transform the host cells of the yeast strain having the lac Z or HIS3 signal gene, which is under the control of the 5′-region of the GAL4 binding sites. The signal gene will be expressed only in those transformed host cells (cotransformants) in which both fusion proteins are expressed and able to interact with each other. When the signal gene is lac Z, the host cells in which it is expressed will turn blue when X-gal is added to the culture. Therefore, the presence of blue colonies indicates that the two studied cloned proteins are able to interact with each other.

Using this digibrid system, the FAS-IC intracellular domain was cloned separately in the pGBT9 vector (carrying the GAL4 DNA binding sequence, obtained from CLONTECH, USA, see below) to obtain a fusion protein with the GAL4 DNA binding domain. To clone FAS-R in pGBT9, a clone encoding the full-length cDNA sequence for FAS-R (see joint application IL 111125) was used, from which the region encoding the intracellular domain of IC was cut using a standard procedure using various restriction enzymes. This clone was then isolated and inserted into the pGBT9 vector, cut with the corresponding restriction enzymes in the region of multiple cloning sites (MCS). It should be noted that FAS-IC occupies a site in the intact FAS-R from 175 to 319 residues, and this FAS-IC site was inserted into the pGBT9 vector (see also IL 111125).

The above hybrid (chimeric) vector, together with cDNA from the HeLa human cell gene library, cloned into the pGADGH vector bearing the GAL4 activation domain were used to co-transfect yeast strain HF7 (all the above vectors are pGBT9 and pGADGH carrying the HeLa cell cDNA library, and also the yeast strain was taken from Clontech Laboratories, Inc., USA, as part of the MATCHMAKER Two-Hybrid System, # PT1265-1).

Co-transfected yeasts were selected for their ability to grow in a medium containing no histidium (His ^- medium), while the growing clones indicated the presence of positive transformants. Selected yeast clones were then tested for the ability to express the lac Z gene, i.e. on the activity of LAC Z in them. This was done by adding X-gal to the culture medium, which, under the action of β-galactosidase, an enzyme encoded by the lac Z gene, decomposes to form a blue product.

Thus, the blue colonies indicate the activity of the lac Z gene in them. For the activity of the lac Z gene, it is necessary that the GAL4 transcription activator is in active form in transformed clones, namely, that the DNA binding domain encoded by the aforementioned hybrid vector is properly connected to the domain activation of GAL4 encoded by another hybrid vector. Such a connection is possible only if two proteins, fused respectively with different GAL4 domains, had the ability to stably interact (bind) with each other. Thus, the isolated His ⁺ and blue (LAC Z ⁺ ) colonies are colonies that are co-transformed with a vector encoding FAS-IC and a vector encoding a protein product from human HeLa cells that can stably bind to FAS-IC.

The plasmid DNA from the above His ⁺ , LAC Z ⁺ yeast colonies was isolated and introduced by electroporation into E. coli strain HB101 using a standard procedure, and then Leu ⁺ and ampicillin resistant transformants were selected, these transformants carrying the pGADGH hybrid vector, which has coding sequences for Amp ^R and Leu ² . Therefore, such transformants are clones carrying sequences encoding newly identified proteins capable of binding to FAS-IC. The plasmid DNA was then isolated from these transformed E. coli cells and tested again as follows:

(a) by retransforming it together with the original hybrid plasmid encoding FAS-R (pGTB9 hybrid carrying FAS-IS) yeast strain HF7, as described above. In the control, co-transformation was performed using vectors carrying sequences encoding an unnecessary protein, for example, pAST-lamin, or only pCtBT9 together with a plasmid encoding a protein that binds FAS-IC (i.e. MORT-1). The co-transformed yeast cells were then tested for their ability to grow in His ^- or in this medium supplemented with various amounts of 3-aminotriazole; and

(c) by retransformation of the yeast host cells of the SFY526 strain with DNA plasmid and the original hybrid plasmid with FAS-IC and control plasmids described in (a), followed by determination of LAC Z ⁺ activity (β-gal formation efficiency, i.e., formation blue colonies).

The results of the above tests revealed that the pattern of colony growth in His ^- medium was identical to the pattern of LAC Z activity, since the tested stained colonies, i.e. His ⁺ colonies also had the LAC Z ⁺ phenotype. Further, LAC Z activity in a liquid medium (preferred culturing conditions) was tested after transfection with hybrid plasmids with a GAL4 DNA binding domain and an activation domain in yeast cells SFY256, which are characterized by better LAC Z inducibility by a GAL4 transcription activator than HF-7 yeast host cells.

Using the above procedure, a protein called HF-1 and now called MORT-1, "Mediator of Receptor-induced Toxicity" was identified, isolated and characterized.

It should also be noted that in a number of the above tests for the expression of β-galactosidase in the digibrid system, the expression of β-galactosidase was tested using a filter test. Screening revealed that 5 of 3 × 10 ⁶ cDNA molecules contained an MORT-1 insert. The cloned MORT-1 cDNA inserts thus isolated were sequenced using standard DNA sequence determination procedures.

The amino acid sequence of MORT-1 was obtained from the nucleotide sequence in DNA. Amino acid residues in the proteins were numbered, as is done in the Swiss-Prot database.

Division mutants were obtained by PCR, and point mutants by oligonucleotide-directed mutagenesis (Current Protocols in Molec. Biol., 1994).

(ii) Induced expression, metabolic labeling and immunoprecipitation of proteins.

MORT-1, N-terminated to the FLAG octopeptide (FLAG-HF-1; Eastman Kodak, New Haven, Ct., USA), FAS-IC chimera, FAS-R, p55-R, including p55R extracellular domain (amino acids with 1 to 168), fused to the transmembrane and intracellular domain of FAS-R (amino acids 153 to 319), and luciferose cDNA used for control were expressed in HeLa cells. Expression was performed using a tetracycline-controlled expression vector in a HeLa cell clone (HtTA-1) in which a tetracycline-controlled transactivator is expressed (Gossen and Bujard, 1992; as described in PCT / US95 / 05854; see also Boldin et al., 1995 ) Metabolic labeling with [ ³⁵ S] -methionine and [ ³⁵ S] -cysteine (DUPONT, Wilmington, De., USA and Amersham, Buckinghamshire, England) was performed 18 hours after transfection for 4 hours at 37 ° C in Eagle’s modified medium Dulbecco, not containing methionine and cysteine, but supplemented with 2% dialyzed fetal calf serum. The cells were then lysed in RIPA buffer (10 mm Tris-HCl, pH 7.5, 150 mM NCl, 1% NP-40, 1% deoxycholach, 0.1% SDS and 1 mM EDTA), and the lysate was pre-clarified by incubation with ballast rabbit antiserum (3 μl / ml) and Sepharose beads with G protein (Pharmacia, Uppsala, Sweden; 60 μl / ml). Immunoprecipitation was performed by incubating for 1 h at 4 ° C 0.3 ml of a lysate with mouse monoclonal antibodies (5 μl / sample) against the FLAG octopeptide (M2; Eastman Kodak), pp 55-P (# 18 and # 20; ( Engelmann et al., 1990)), or FAS-R (ZB4; Kamiya Southand Oaks, Ca., USA), or with the corresponding isotypic mouse antibodies as a control, followed by incubation for 1 h with Sepharose beads loaded with G protein .

(iii) In vitro Binding

Glutathione S-transferase was prepared, fused with wild-type Fas-IC or mutated Fas-IC, and adsorbed onto glutathione beads (as described in IL 109632, 111125, 112002, 112692; see also Boldin et al., 1995; Current protocols in molecular biology, 1994; Frangioni and Neel, 1993). The binding of the metabolically labeled FLAG-HF-1 fusion protein to CST-Fas-IC was evaluated by incubating the beads for 2 hours at 4 ° C with HeLa cell extracts metabolically labeled with [ ³⁵ S] methionine (60 μC / ml), which express FLAG-HF-1. The extracts were prepared in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 1 mM dithiotriethol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 20 μg / ml aprotonin, 20 μg / ml of leupeptin, 10 mm sodium fluoride and 0.1 mm sodium vanadate (1 ml per 5 × 10 ⁵ cells).

(iv) Analysis of cytotoxicity triggered by induced expression of HF-1.

HF1, Fas-IC, p55-IC, and luciferase cDNAs were inserted into a tetracycline-controlled expression vector and transfected HtTA-1 cells (HeLa cell line) (Grossen and Bujard, 1992) together with secreted alkaline placenta phosphatase cDNA controlled by the SV40 promoter (pSBC-2 vector ((Dirks et al., 1993)). Cell death was assessed 40 hours after transfection in a neutral red cell uptake test (Wallach, 1984) or to specifically assess the death of those cells that express transfected cDNA by determine the amount of alkaline phosphatase from p atsenty secreted into the growth medium at the 5th hour incubation.

In another series of experiments, to analyze the region of the MORT-1 protein (HF-1) involved in binding to FAS-IC, HeLa cells that contain a tetracycline-controlled transactivator (HtTA-1) induced transient expression of the following proteins using tetracycline-controlled expression vector (pUHD 10-3): human FAS-R only; FAS-R, as well as the N-terminal portion of MORT-1 (amino acids 1 to 117, "head portion of MORT-1"); Human FAS-R, as well as the C-terminal portion of MORT-1, which contains a homologous region of its "death domain" (amino acids 130 to 245, "MORT-100", see also IL 112742); FLAG-55.11 (amino acids 309-900 of protein 55.11, fused at its N-terminus with the FLAG octopeptide, wherein 55.11 is a specific binding protein p55-IC, see also IL 109632). 12 hours after transfection, cells were harvested by trypsinization and re-seeded at a concentration of 30,000 cells / cell. After incubation for 24 hours, the cells were treated for 6 hours with monoclonal antibodies against the extracellular domain of FAS-R (monoclonal antibodies CH-11, Oncor) at various concentrations (0.001-10 μg / ml monoclonal antibodies) in the presence of 10 g / ml cycloheximide. After this, the cell viability was determined in the neutral red absorption test and the results were calculated in% of living cells relative to the number of cells incubated with one cycloheximide (in the absence of monoclonal antibodies CH-11 against FAS-R).

(v) Northern analysis and sequencing.

Poly A ⁺ RNA was isolated from HeLa cells (Oligotex-dT mRNA kit, QIAGEN, Hilden, Germany). Northern analysis using HF-1 cDNA as a sample was performed using the conventional method (as described in PCT / US95 / 05854; see also Boldin et al., 1995). The nucleotide sequence for MORT-1 (HF-1) was determined in both directions using the dideoxy chain termination method.

In the above experiments, the following results were obtained, shown in Table 1 and Figure 1-7. Sequence analysis of the MORT-1 cDNA cloned by the digibrid procedure showed that it encodes a new protein (see below).

The use of a hybrid test to evaluate the binding specificity of this protein (MORT-1, "Mediator of Receptor-induced Toxicity") with Fas-IC and to determine the specific region in Fas-IC with which it binds led to the following results (Table 1) :

(a) this protein binds to both human and murine Fas-IC, but does not bind to a number of other tested proteins, including three receptors from the TNF / NGF family of receptors (TNF receptors p55 and p75 and CD40);

(b) it is shown that the substitution mutation at position 255 (Ile) in the FAS-R “death domain” blocking signal transmission both in vitro and in vivo (1pr ^cg mutation (Watanabe-Fukunaga et al., 1992; Itoh and Nagata, 1993)), also prevents the binding of MORT-1 to FAS-IC;

(c) the MORT-1 binding site in FAS-R is in the “death domain” of this receptor, and

(d) MORT-1 communicates with itself. Different regions of the protein are involved in self-association and in the binding of HF-1 to FAS-R. The MORT-1 fragment corresponding to residues 1 to 117 binds to the full-sized MORT-1 molecule, but does not bind to either the same fragment or FAS-IC. Conversely, the fragment corresponding to residues 130 through 245 binds to FAS-R, but does not bind to MORT-1. In addition, from the results shown in Table 1, it is seen that the FAS-R death domain region is critical for FAS-IC self-association, as is the p55-R death domain region for p55-1C self-association. Deletions on both sides of this “death domain” do not affect their ability to self-associate, however deletions within these “death domains” affect self-association.

In the case of MORT-1, the binding of MORT-1 to FAS-IC also depends on the whole (full-sized) FAS-R death domain, the binding to FAS-R is independent of regions outside the FAS-R death domain.

Table 1 shows the results of the interaction of pairs of proteins, one of which is encoded by the pCBT9 construct, which also contains the code for the GAL4 protein binding domain, and the other is encoded by the pGAD-CH construct, which also contains the GAL4 activation domain code. The interaction of these proteins took place in yeast cells SF 526, co-transfected with the indicated constructs, and was analyzed using a filter test for β-galactosidase expression. The DNA binding domain constructs include four human Fas-IC constructs; four mouse Fas-IC constructs comprising two full-length constructs having mutations of the replacement of Ile by Leu or Ile by Ala at position 225 (I225N and I225A, respectively); and three designs of HF-1 (MORT-1). All these constructs are shown on the left side of Table 1. The activation domain constructs include three HF-1 constructs, the constructs of the HF-1 parts being the same as the constructs of the DNA binding domain; the construction of full-length human Fas-IC, wherein the construction of the Fas-IC portion is the same as the above construction of the DNA binding domain. The intracellular domains of the human p55 receptor TNF (residues 206-426 p55-IC), human CD40 (residues 216-227) and the human TNF p75 receptor (residues 287-461), as well as the lamin, cyclin D and the empty Gal4 vector (pGBT9 ) served as negative controls in the form of DNA-binding domain constructs. SNF-1 and SNF4 served as positive controls in the form of DNA binding domain (SNF1) and activation domain (SNF4) constructs. The empty GAL4 vectors (pGADCH) also served as negative controls in the form of activation domain constructs (for more details on p55-IC and p75-IC see PCT / US95 / 05854). The symbols "++" and "+" indicate the development of a strong color during 30 and 90 min of the test, respectively; and “-” means no staining for 24 hours. Combinations for which these symbols are absent were not tested.

Expression of HF1 (MORT-1) molecules fused to the N-terminus of the FLAG octopeptide (FLAG-HF-1) resulted in the formation of HeLa cells in four different sizes of proteins: about 27, 28, 32 and 34 kD. Figure 1 (A and B) shows the results demonstrating the interaction of HF1 with Fas-IC in vitro. As indicated above in the description of FIGS. 1A and B, FIG. 1 reproduces a control radio-autograph of protein immunoprecipitate from HeLa cell extracts transfected with a FLAG-HF1 fusion protein (FLAG-MORT1) or luciferase cDNA as a control. 1B, a radio autograph is shown showing the in vitro interaction between HF1 and Fas-IC, where HF1 is in the form of [ ³⁵ S] methionine-labeled HF1-FLAG fusion protein obtained from HeLa transfected cell extract, and Fas-IC in as human and mouse fusion proteins GST-FAS-IC, one of which has a substitution mutation at position 225 in Fas-IC. All of these GST-FAS-IC fusion proteins were obtained in E. coli cells. GST fusion proteins were attached to glutathione beads prior to reaction with extracts containing the HF1-FLAG fusion protein. After this interaction, SDS-PAGE was performed. Thus, in vitro interactions were evaluated by autoradiography after SDS-PAGE to bind the metabolically labeled HF1 protein synthesized in HeLa cells as part of the HF1 fusion protein with the FLAG octopeptide (FLAG-HF1), with GST fused to human or mouse Fas-IC (GST-huFas-IC, GST-mFas-IC); or with a GST fused to Fas-IC containing a mutation of the replacement of Ile with Ala at position 225. As can be seen from Figure 1B, all four FLAG-HF1 proteins show the ability to bind to Fas-IC upon incubation with the GST-Fas-IC fusion protein . As in the test in the yeast digibrid system, HF1 does not bind to the GST-Fas-IC fusion protein containing the lpr ^cg mutation site (I225A).

Proteins encoded by FLAG-HF1 cDNA also show the ability to bind to the intracellular domain of FAS-R, as well as to the chimeric intracellular domain of FAS-R, in which the intracellular domain is replaced by the corresponding p55-R domain (p55-FAS) under conditions of joint expression of these receptors in HeLa cells. Figure 2 (A, B, C) shows the results demonstrating the interaction of HF1 with FAS-IC in transfected HeLa cells, i.e. in vivo. As mentioned above, in the description of FIGS. 2 A, B, C, these autographs reproduce precipitate autographs from various transfected HeLa cells that demonstrate in vivo interaction and the specific interaction between HF1 and FAS-IC in cells co-transfected with constructs encoding these squirrels. Thus, the FLAG-HF1 fusion protein was expressed and metabolically labeled for [ ^35- S] -cysteine (20 μC / ml) and for [ ³⁵ S] -methionine (40 μC / ml), in HeLa cells separately or together with the human FAS-R chimera, FAS-R, in which the extracellular domain of FAS-R is replaced by the corresponding region of human p55-R (p55-FAS), or with p55-R as a negative control. Cross-immunoprecipitation of HF1 with a co-expressed receptor was performed using these antibodies (Fig. 2A-C). 2A-C, FLAG-HF1 is able to bind to the intracellular domain of FAS-R, as well as to the chimeric intracellular domain of FAS-R-p55-R, having the extracellular domain of p55-R and the intracellular domain of FAS-R under conditions of joint expression of these receptors in HeLa cells (see middle tracks of FIG. 2A and three tracks to the left in FIG. 2C, respectively). Further, immunoprecipitation of FLAG-HF1 from extracts of transfected cells also leads to precipitation of co-expressed FAS-R (Fig. 2A) or co-expressed p55-FAS chimera (Fig. 2C). Conversely, immunoprecipitation of these receptors leads to joint precipitation of FLAG-HFl (Figa and 2C).

Northern hybridization analysis using HF1 cDNA as a probe reveals one hybridizing transcript in HeLa cells. Figure 3 shows Northern blotting in which poly A ⁺ PHK (0.3 μg) from transfected cells was hybridized with HF1 cDNA. The size of this transcript (about 1.8 kV) is close to the size of the HF1 cDNA (about 1702 nucleotides).

Sequence analysis revealed that this cDNA contains an open reading frame for approximately 250 amino acids. Figure 4 shows the preliminary nucleotide sequence and the HF1 amino acid sequence obtained from it, in which the main part of the "death domain" is underlined in the same way as the possible starting Met residue (position 49, bold M), and the translation stop codon is marked ( asterisk under the codon at position 769-771). The main part of the “death domain” reveals homology with the known main parts of the “death domains” p55-R and FAS-R (p55 DD and FAS-DD). To accurately determine the C-terminal portion of HF-1 and obtain accurate data on the N-terminal portion of HF1 (starting residue Met), the following experiments were performed.

Using the methods described above, a number of constructs were constructed that encode HF1 molecules fused at their N-ends to the FLAG octopeptide (FLAG-HF1). When these constructs were expressed in HeLa cells, the expressed proteins were metabolically labeled using ³⁵ S-cysteine and ³⁵ S-methionine (see above for the same in Fig. 5B). HF1-FLAG molecules were encoded by the following cDNAs containing different regions with a code of different regions H 1:

i) FLAG octopeptide cDNA attached to the 5′-end of the HF1 cDNA from which nucleotides 1 through 145 are excised (see FIG. 4);

ii) the FLAG octopeptide cDNA attached to the 5′-end of the full length HF1 cDNA (see the FLAG-HF1 construct shown above in FIG. 1B);

iii) the FLAG octopeptide cDNA attached to the 5′-end of the HF1 cDNA from which nucleotides 1 through 145 and nucleotides 832 through 1701 (see FIG. 2) are excised and the GCC codon at position 142-144 is changed to TCC in order to prevent the start of broadcasting on this site.

After expression of the above HF1-FLAG fusion products, they were immunoprecipitated as described above using either anti-FLAG monoclonal antibodies (M2) or, in control, anti-p75TMF-R antibodies (H9), and then separation into SDS-PAGE ( 1% acrylamide) and radioautography. The results are shown in FIG. 5, which reproduces a radio autograph of the separated above fusion proteins, samples of which were applied to the gel tracks, as indicated below.

Lanes 1 and 2: HF1-FLAG fusion protein encoded by the FLAG octopeptide cDNA attached to the 5 ′ end of the HF1 cDNA from which nucleotides 1-145 were excised.

Lanes 3 and 4: HF1-FLAG fusion protein encoded by the FLAG octopeptide cDNA attached to the 5′-end of the full-length HF1 cDNA.

Lanes 5 and 5: the HF1-FLAG fusion protein encoded by cDNA attached to the 5′-end of the HF1 cDNA from which nucleotides 1-145 and 832-1701 were cut, and the GCC at position 142-144 was replaced by TCC so that to prevent the start of broadcasting on this site.

Immunoprecipitation of samples in lanes 2, 4, and 6 was performed with anti-FLAG monoclonal antibodies, and samples in lanes 1, 3, and 5 with anti-p75TNF-R antibodies.

From the radio autograph in FIG. 5, the following is seen. The identity of the product sizes on lanes 2 and 4 confirms that nucleotides 769-771 are translation termination sites for HF1, i.e. this codon is a stop signal, as shown by an asterisk in FIG. 4. Further, the presence of a wide band, which actually contains two translation products (visible on the gel, but due to strong tagging merging into one large band on the radio autograph) on track 6, shows that the presence of two additional products (higher molecular weight in broad bands) in lanes 2 and 4 reflects the fact that translation initiation occurs both at the N-terminus of the fusion molecule and from the methionine residue at position 49 in the HF-1 sequence (see boldly underlined M at position 49 of the amino acid sequence atFigure 4). Thus, the above results confirm the correctness of the reduced amino acid sequence of the C-terminus of HF1 and serve as evidence that the N-terminus of HF-1 can be located at position 49 of the sequence depicted in FIG. 4.

Indeed, in additional experiments on the expression of HF-1 without the FLAG octopeptide attached to its 5′-end, it was shown that Met ⁴⁹ serves as an effective translation initiation site.

It should be recalled that when searching the “Gene Bank” and “Protein Bank” data banks, sequences corresponding to the HF-1 sequences shown in FIG. 4 were not found. Thus, HF1 is a new specific binding protein FAS-IC.

A high level of p55-IC expression triggers a cytotoxic effect (see IL 109632, 111125 and Boldin et al., 1995). Expression of Fas-IC in HeLa cells also leads to this effect, although smaller in magnitude, which can only be detected using a sensitive test. 6A and B are diagrams for ligand-independent triggering of cytotoxic effects in cells transfected with HF1 as well as human p55-IC and FAS-IC. The effect of the transient expression of human HF1, Fas-IC, human p55-IC, or luciferase — in the control — on the viability of HeLa cells was analyzed using an expression vector controlled by tetracycline. Cell viability was determined 40 min after cDNA administration in the presence (unstained columns of Figs. 6A and B) or in the absence (shaded columns of Figs. 6A and B) of tetracycline (1 μg / ml, for expression block) together with cDNA encoding secreted alkaline placental phosphatase. Cell viability was determined either in the neutral red dye uptake test (Fig. 6A) or in a specific determination of the viability of cells that express transfection DNA by measuring the amount of alkaline phosphatase of the placenta secreted into the growth medium (Fig. 6B).

6A and B show that significantly more cells die in the expression of HF1 in HeLa cells than in the expression of FAS-IC. The cytotoxic effects of p55-IC, FAS-IC, and HF1 are apparently associated with the regions of the “death domain” that are present in all of these proteins, whose “death domains” are predisposed to self-association and, possibly, therefore, provoke cytotoxic effects. The above properties of HF1 (MORT-1); namely, the specific association of HF1 with a particular region in FAS-R, which is involved in the induction of cell death, and the fact that even a small structural change in this region (mutation lpr ^cg ), which prevents signal transmission, also blocks HF1 binding, show that this protein plays a role in signaling and triggering cell death. This conclusion is confirmed by the discovered ability of HF1 to trigger the effect of intrinsic cytotoxicity of the cell. So, HF1 (MORT-1) can function (i) as a modulator of FAS-R self-association due to its ability to bind to FAS-R in the same way as in self-association, or (ii) serve as a docking point for other proteins that are involved in transmission FAS-dependent signals, i.e. HF1 can be a docking protein and therefore bind other receptors along with FAS-R, or (iii) is an integral part of another signaling system that interacts with the FAS-R signaling system.

To further analyze the MORT-1 (HF1) regions involved in FAS-R binding and modulation of the cellular effects (cytotoxicity) mediated by FAS-R, the above experiments were performed using vectors encoding the MORT-1 proteins ("head of MORT- 1 ", amino acids 10117 and" MORT-1 dd ", amino acids 130-245) (separately), with a vector encoding FAS-R, for co-transfection of HeLa cells. In these experiments, temporary expression of various proteins and protein combinations was performed in HeLa cells that contain a tetracycline-controlled transactivator (HtTA-1) by inserting sequences encoding these proteins into a tetracycline-controlled pUHD 10-3 expression vector. In control transfections, vectors encoding only FAS-R and vectors encoding the FLAG-55.11 fusion protein were used (protein 55.11, which is a portion of the protein from amino acids 309 to 900 that specifically binds p55-IC, was fused (its N-terminus) with octapeptide FLAG).

Transfected cells after appropriate incubation periods (see (iv) above) were treated with various concentrations of anti-FAS-R monoclonal antibodies (CH-11), which specifically bind to the FAS-R extracellular domain expressed by the cells. Such anti-FAS-R antibody binding induces FAS-R aggregation on the cell surface (much the same as when binding to the FAS-R ligand) and induces a chain of intracellular signals mediated by FAS-IC, which ultimately leads to cell death ( cell cytotoxicity mediated by FAS-R). Used concentrations of monoclonal antibodies anti-FAS-R (CH-11) 0.01-10 μg / ml, usually such concentrations of 0.005; 0.05; 0.5 and 5 μg / ml. Cells were treated with anti-FAS-R antibodies in the presence of 10 μg / ml cycloheximide.

The results of the above analysis are plotted in FIG. 7, in which% of viable transfected cells is shown as a function of the concentration of anti-FAS-R monoclonal antibodies (CH-11) used to treat the cells for each of the four groups of transfected cells. These groups of transfected cells are indicated by different symbols: (i) open squares indicate cells transfected only with a control vector encoding the FLAG-55.11 fusion protein ("55.11", negative control) that does not bind FAS-IC; (ii) filled squares denote cells co-transfected with vectors encoding FAS-IC and vectors encoding the C-terminal portion of MORT-1, amino acids 130-245, which contains a region homologous to the "death domain" (dd) MORT-1 (" fas + mort Idd "); (iii) filled triangles indicate cells transfected only with a vector encoding FAS-R ("fas", positive control); and (iv) open circles indicate cells co-transfected with vectors encoding FAS-R and vectors encoding the N-terminal portion of MORT-1, amino acids 1-117, “MORT-1 head” (“fas-morthe”).

From the results presented in Fig. 7, it is seen that the expression of FAS-R in transfected cells gives the cell increased sensitivity to the cytotoxic effect of anti-FAS-R antibodies (compare "fas" with "55.11"). Further, co-expression of the MORT-1 region, which contains a region homologous to the "death domain" and FAS-R ("fas + mort Idd"), strongly inhibits the death of a cell induced by FAS (ie, mediated by FAS-R), as this was to be expected based on the ability of the region of the “death domain” DD MORT-1 to bind to the “death domain” FAS (FAS-DD). In addition, the co-expression of the N-terminal portion of MORT-1 and FAS-R ("fas + morthe") either does not affect cell death mediated by FAS-R at all, or only slightly increases cytotoxicity (i.e. slightly increases cell death )

Thus, the above results clearly show that the MORT-1 protein has two different regions involved in FAS-IC binding and mediation of the cell cytotoxic activity by the FAS-IC domain.

Therefore, these results provide the basis for using different sites (i.e., active fragments or analogues) of the MORT-1 protein in various pharmaceutical applications. For example, those analogues or fragments or derivatives of the MORT-1 protein that mainly contain only the C-terminal portion of MORT-1, including its "death domain", can be used to suppress FAS-R-mediated cytotoxic effects in cells or tissues containing FAS-R, and protecting, in this way, these cells or tissues from the harmful effects of the FAS-R ligand, for example, in cases such as acute hepatitis. Conversely, those analogs or fragments or derivatives of the MORT-1 protein that mainly contain only the N-terminal portion of MORT-1 can be used to increase FAS-R mediated cytotoxic effects in cells or tissues containing FAS-R and to enhance in this way, the destruction of these cells or tissues in those cases when it is desirable, for example, tumor cells or autoreactive T and B cells. The above applications of different regions of MORT-1 can be accomplished, as indicated above, by using various recombinant viruses (e.g., from smallpox) to embed various sequences encoding the MORT-1 regions into specific cells or tissues that need treatment.

In addition, you can also get and use other various molecules, for example antibodies, peptides and organic molecules, the sequences and molecular structures of which correspond to the above MORT-1 sites, to achieve the same desired effects that are mediated by these MORT-1 sites.

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Claims (12)

1. A DNA molecule encoding a polypeptide capable of binding to the intracellular domain of the FAS ligand receptor (FAS-IC), including: (1) the nucleotide sequence of SEQ ID No: 1 protein MORT-1; (2) a nucleotide sequence that encodes an analog of the indicated MORT-1 protein, wherein said analogue differs from the MORT-1 protein in a single amino acid residue and binds to FAS-IC; (3) a nucleotide sequence consisting of a nucleotide sequence that encodes a fragment of said MORT-1 protein, wherein said protein fragment binds to FAS-IC; or any of the variants of the nucleotide sequence indicated in (1), (2) or (3) obtained on the basis of the degeneracy of the genetic code. 2. The DNA molecule according to claim 1, characterized in that it has a DNA sequence encoding the amino acid sequence of SEQ ID No: 2. 3. The DNA molecule according to claim 2, characterized in that it has a DNA sequence of SEQ ID No: 1. 4. A polypeptide capable of binding to the intracellular domain of FAS-R encoded by the DNA according to claim 1. 5. The polypeptide according to claim 4, characterized in that it has the deduced amino acid sequence of SEQ ID No: 2. 6. A vector for expression in eukaryotic cells of a polypeptide that binds to the intracellular domain of FAS-R, including the DNA sequence according to one of claims 1 to 3, operably linked to regulatory sequences. 7. A method of producing a polypeptide that binds to the intracellular domain of FAS-R, comprising growing host cells transformed with the vector according to claim 6, under conditions ensuring the expression of the polypeptide, effective post-translational modification and its isolation. 8. A method of modulating the action of a FAS-R ligand on cells bearing FAS-R, comprising treating said cells with a polypeptide according to claim 4 or 5, capable of binding to the intracellular domain of FAS-R and modulating the activity of FAS-R. 9. A method of modulating the effect of a FAS-R ligand on cells, comprising applying a procedure using a ribozyme in which a vector encoding a ribozyme sequence capable of interacting with a cell mRNA sequence encoding a polypeptide according to claim 4 or 5 is introduced into said cells in the form providing expression of the indicated ribozyme sequence in these cells, and when expressing the specified ribozyme sequence in these cells, it interacts with the specified sequence of cellular mRH and cleaves said mRNA sequence resulting in the inhibition of expression of MORT-1 in said cells. 10. A pharmaceutical composition for modulating the action of a FAS-R ligand on cells, comprising, as an active ingredient, the polypeptide of claim 4 or 5, or a mixture thereof. 11. A pharmaceutical composition for modulating the action of a FAS-R ligand on cells, comprising as an active ingredient an animal virus recombinant vector carrying a sequence encoding a protein capable of binding to a cell surface receptor and a sequence encoding a polypeptide according to claim 4 or 5. 12. A method for isolating and identifying a protein capable of binding to the intracellular domain of FAS-R, comprising applying the Southern hybridization procedure under non-stringent conditions, followed by cloning by PCR, in which the sequence or part thereof according to any one of claims 1 to 3 is used in as a probe that binds to sequences from a cDNA library or genomic DNA having at least partial homology with a probe, the above linked sequences being then amplified and cloned using the PCR procedure to obtain clones encoding proteins having at least partial homology with the indicated sequences according to claims 1-3.

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