UA76933C2 - Dna molecule that codes the polypeptide that binds with the p55 domain of a tnf receptor; protein and a method for generating it - Google Patents

Abstract

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

Description

Description of the invention

The present invention relates to receptors belonging to the TME/MOBE receptor superfamily, and 2 to the regulation of their biological functions. The superfamily of TME/YUMOE receptors includes tumor necrosis factor receptor p55 and p75 (TME-K), receptor ligand RAZ (also called EAZ/APO1 or

EAZ-K, hereinafter referred to as RA5B-K) and other receptors. More specifically, the present invention relates to

Kk new proteins that bind to the intracellular domains (1C) of p55- and p75-TME-K and Rav-K, (hereinafter referred to as p5B1C, p7/751C and BRaz-1C, respectively), and which have the ability to modulate the functions of p5b5B- and p75-TME receptors, as well as the Raz receptor. One of such proteins capable of binding to ro51C of intact ro5-TME-K is ro51C itself in the form of the ro51C molecule or its part, for example, the so-called "death domain" ro551C. Thus, the present invention also relates to new TME-associated effects that can be induced in cells by the intracellular domain of p5B5-TME (p551C) or its part in a ligand (TME)-independent manner. In addition, the present invention relates to the preparation and use of novel proteins that bind to rBB- and p75-TME-K, and proteins that bind to Raz-K, which are described herein as ro51c-, p751C- and Raz-1C- binding proteins.

In another aspect, the present invention also relates to new soluble oligomers! TMRE-K, oligomeric BAB-K and oligomeric receptors, which are a mixture of TME-K and BAB-K; to the use of these receptors, and to methods of their production.

Tumor necrosis factor (TME-o) and lymphotoxin (TME-B) (hereinafter referred to as TME-zh and TME-D, respectively) are multifunctional cytokines produced, mainly, by mononuclear phagocytes, and possessing multiple effects on the cell (MMaiyasi, O (1986), in

Ipiepegop 7 (op Ogezzeg, eo.), years 83-122, Asadetis Rgez5, Gopdop; and Veshieg 5 Segati (1987)). Action with 29 of both indications of cytokines, TME-o and TME-Bd is initiated by means of their binding to specific Ge) receptors on the cell surface. It is obvious that some actions of these cytokines have a beneficial effect on the body, that is, they can, for example, destroy tumor or virus-infected cells and enhance the antibacterial activity of granulocytes. In this case, TME provides protection of the body against tumors and infectious agents, and also promotes wound healing. Thus, TME can be used as an antitumor agent that binds to its receptors on the surface of tumor cells and thereby initiates events leading to the death of those tumor cells. TME can also be used as an anti-infective agent. F

However, both factors, i.e., TME-o; and TME-rd also have an adverse effect. It has been shown that the so-called overproduction of TME-5 can play an important role in the pathogenesis of some diseases. So, for example, it is already known that the effect of TME-o, mainly on the vascular network of the organ, is a symptom! of septic shock |Tgaseu ei a!., 1986). In some diseases, TME can also cause a sharp decrease in body weight (cachexia) as a result of suppressing the activity of adipocytes and provoking anorexia, and then the factor of tumor necrosis of TME is also called cachexia. Veshshchieg 5 Segati, in p. 1987), and is the main mediator of rejection observed in "transplant versus host" reactions and IRidney et al. 1987). In addition, it is known that TME participates in the process of inflammation and mediates many other "" diseases.

Two different and independently expressed p55- and p/5-TME-K receptors, which specifically bind to TME-o and TME-rD, initiate and/or mediate the biological functions of TME for various indications. These two receptors have a structurally different intracellular domain, which suggests that they transmit a different signal! See Noppitap et al., 1989; EpdeItapp et al., 1990;

Fo KotosKpaiz et al., 1990; I eoivspeg et al., 1990; Zspaii et al., 1990; Norpag et al., 1990; Tiy et al., 1990; and (Se) Neieg et al., 1990). However, the cellular mechanisms, for example, various proteins and possibly other factors involved in the transmission of the intracellular signal of r75-TME-K, have not yet been identified! (new proteins capable of binding to p751C and p551C are described below for the first time). But it is precisely this kind of signal transmission, which occurs casually after the binding of a ligand (that is, TME-o or TME-p) to a receptor, that is responsible for the initiation of a cascade of reactions, which, in the end, lead to the observed cellular response to TME.

As for the above-mentioned cytocidal action of TME, in most cells studied to date, the action is stimulated, mainly, by the p55-TME receptor. Antibodies against the extracellular domain (ligand-binding domain) of p5-TME-K can act as stimulants by themselves

IF) of the cytocidal effect | see, EP 4124861, which correlates with the effectiveness of cross-linking of the receptor with antibodies, and which is, obviously, the first stage of the intracellular signal transmission process. In addition, studies carried out by the mutation method IVgaskerisy et al., 1992, Tapadiya et al., 60 1993) showed that the biological function of p55-TME-K depends on the integrity of its extracellular domain, which gives grounds to assume that the initiation of intracellular transduction the signal that induces the cytocidal effect of TME occurs as a result of combining two or more intracellular domains of p5-TME-K. In addition, TME (o; i p) is present as a homotridimer and, as it has been suggested, induces p55-TME-K-mediated intracellular signaling due to its ability to 65 bind and cross-link with receptor molecules, i.e., To create aggregation with the receptor.

Below, in this application, it will be described how rbo51cS and ro55OrO can associate with each other,

and induce a ligand-independent way, TME-association effect! in cells

The second member of the TME/MOGR receptor superfamily is the RAB receptor (HA5B-K), which is called the Raz antigen, and which is a cell surface protein expressed in various tissues and has homology with a number of cell surface receptors, including TME-K and

MOB-K, RAB-K mediate the death of cells by the type of apoptosis (Mop et al., 1991), and apparently serve as a negative selector of autoreactive T-cells, i.e. in the process of maturation of T-cells, RAB-K mediates apoptosis of T-cells that recognize autoantigens. It was also found that mutations in the RA5B-K (1rg) gene cause a violation of lymphoproliferation in mice, which have a similar to the human picture of such an autoimmune disease as systemic lupus erythematosus (SLE) (MUaiapare-Rykypada et al., 1992) . The ligand for EAB-K is, obviously, a molecule associated with the cell surface and carried, among others,

by killer T-cells (or cytotoxic T-lymphocytes - STIs), and then when such STIs. come in contact with cells carrying ЕГА5-К, they are capable of inducing apoptosis of ГА5-К-bearing cells. In addition, a monoclonal antibody with specificity for BAB-K was obtained, which had the ability to induce apoptosis of cells carrying RABZ-K, including muscle cells, transformed with cDNA encoding human EA5B-K |op et al., 19911.

It was also found that, in addition to T-lymphocytes, there are other normal cells that express EBAB-K on their surface and that can be destroyed by stimulating this receptor. It was suggested that the unregulated induction of this process of cytolysis leads, in some diseases, to the destruction of body tissues, for example, to the destruction of liver cells in acute hepatitis. Therefore, the development of ways to limit the cytotoxic activity of BAB-K can lead to the development of important therapeutic methods.

And vice versa, since it was also found that some malignant cells and

HIV-infected cells carry EA5-K on their surface, so antibodies against EAB-K or GAZ-K-ligand can be used to stimulate RAZ-K-mediated cytotoxic effects, which will allow obtaining a means of destroying malignant or HIV-infected cells cells (see, Mop et al., and) 1991). In addition, obtaining other methods of enhancing the cytotoxic activity of RAB-K may also have important therapeutic value.

The need to obtain a method of modulating the cellular response to TMP (o or p) with EAZ-K ligand has long been ripe. So, for example, in the pathological processes mentioned above, where there is an oversynthesis of TME or PHA5-K-ligand, it is desirable to inhibit TME- or HA5-K-ligand-induced cytocidal effect; and and in other situations, for example, with a healed wound, it is desirable to stimulate the action of TME, or, in the case of tumor or HIV-infected cells, it is desirable to stimulate the EAZ-K-mediated response.

The authors of the present invention have developed several methods (see, for example, application for it, European patent EP 186833, EP 308078, EP 398327 and EP 412486) of regulating undesirable effects of

TME by inhibiting the binding of TME to its receptors with the help of antibodies against TME or by using soluble TME receptors (which are mainly soluble extracellular domains of receptors) to compete for the binding of TME to TME receptors associated with the cell surface. In addition, based on the fact that in order to obtain a TME-induced cellular response, it is necessary to bind TME to its receptors, the authors of the present invention were able to develop a method. for example, ERO 568925)| regulation of TME action by modulating the activity of TME receptors. In particular, in ERO Mo 568925) a method of modulating signal transduction and/or hydrolysis in TME-K is described, as a result of which peptide or other molecules can interact either with the receptor itself or with effector proteins interacting with the receptor, carrying out by the same token, the modulation of the normal functioning of TME-K. In ERO 568925, the construction and characterization of various mutant ro5-TME-receptors with mutations in the extracellular, transmembrane, and intracellular domains of ro5-TME-K are also described. Thus, areas

Fos located in the above-mentioned domains of ro55-TME-K were identified as the main areas

Ge) functioning of the receptor (that is, binding to the TME ligand) with subsequent signal transduction and intracellular signal transmission, ultimately leading to the TME effect observed on cells. In addition, this patent describes a number of methods for the isolation and identification of proteins, peptides, and other se» factors, which are capable of! bind to different areas in the TME-K multi-indication domains, and in addition, the indication of proteins, peptides and other factors! may participate in the regulation or modulation of TME-K activity.

IN IERO 568925| the methods of isolation and cloning of DNA sequences, 5v5, encoding the instructions of proteins and peptides; methods of constructing expressing vectors for the production of these proteins and peptides; and methods of obtaining antibodies or their fragments, which iF) interact with TME receptors or with multi-specific proteins and peptides that bind to different regions of TNE-K. However, in (ERO 568925) there are no descriptions of the proteins and peptides of the present invention that bind to the intracellular domains of TME-K (for example, p55-TME-K), and there are also no descriptions of the method of isolation and identification of such proteins or peptides using yeast two-hybrid systems! In addition, the descriptions have not been written yet! proteins or peptides capable of binding to the EA5B-K intracellular domain.

Thus, to inhibit the action of TME or RA5Z-K-ligand, it is necessary to reduce the amount or activity of TME-K or EBA5B-K on the cell surface, while to stimulate the action of TME or 65 EAZ-K-ligand, it is necessary to increase the amount or activity of TME- K or GA5Z-K. For this purpose, recently, the authors of the present invention were sequencing and analyzing promoters! p5oB5-TME-K and p75-TME-K, as a result of which it was established that a number of their key "motifs" have sequences characteristic of various transcription regulation factors; and therefore, in fact, the expression of TME-K can be regulated at the level of their promoter, i.e., inhibition of transcription can be achieved,

Promoter-initiated, in order to reduce the number of receptors, or enhance transcription, initiated by the promoter, in order to increase the number of receptors (see, 1 104355 and 11 .109633, and their corresponding, not yet published European patent and PCT patent)| Corresponding studies related to the regulation of RHA5-K at the level of the GAZ-K gene promoter have not yet been reported.

In addition, it should also be noted that although, as is known, the receptor! tumor necrosis factor (TME) and their 7/0 structurally related receptor BAB-K (stimulator in cells), after stimulation by leukocyte-producing ligands, show destructive activity, which leads to their own destruction, however, the mechanism! such stimulation is still poorly understood. Mutational studies have shown that EAB-K and P5O5-TME-receptor (p55-K) transmit the signal! cytotoxicity, which have special regions in their intracellular domains | Vgaskeryzi et al., 1992; Tapadiia et al., 1993; op and Madaiya, 75 ..1993). These domains ("domain! of death") have a similar sequence. Such "domains of death" of both GA5-K and ro5-K have a tendency to self-associate. Self-association of these domains apparently stimulates the aggregation of these receptors, which is necessary for the initiation of signal transmission (as shown below, as well as

Yisak is described by Zopd et al., 1994; MUayasy and others; 1994; Voidip et al., 1995), and at high levels of receptor expression can lead to stimulation of ligand-independent signal transmission (as shown below and as described by Voidip et al., 1995).

Thus, prior to the present invention, there were no reports of the production of proteins that can regulate the action of ligands belonging to the TME/MOE superfamily (for example, the action of TME or RGAZ-K-ligand on cells), mediating intracellular signal transduction, controlled , most likely, by the intracellular domains (1C) of receptors belonging to the superfamily of TMP/MOP receptors (such as TME receptors)), namely, by the intracellular domains of ro5- and p75-TME-k (ro51cC and p751C, respectively) ), as well as EA5-1C. and)

Accordingly, one of the goals of the present invention is to obtain proteins capable of binding to the intracellular domains of TME-K and RAB-K, and these proteins, as it is currently assumed, participate in intracellular signal transmission initiated by binding to TME with ego receptors, or binding of RAZ-ligand with ego receptors.

Another goal of the present invention is to obtain antagonists (for example, antibodies) against the indicated proteins that bind to the intracellular domain (1C-binding antibody), which, if necessary, can be used to inhibit signal transmission, in this case, if such 1C - binding proteins are positive signal effectors (i.e., induce signal transmission), ee, or they can be used! to enhance signal transmission, this is the case if such 1C-binding proteins are negative signal effectors (i.e., inhibit signal transmission).

Another goal of the present invention is the use of such 1C-binding proteins for the isolation and characterization of other proteins or factors that can, for example, participate in subsequent signal transmission processes, and/or for the isolation and identification of other receptors "participating in the preceding signal transmission process" , with which these 1C-binding 8 c proteins bind (for example, other TME-K or related receptors/), and therefore, for the functions of which these proteins are also responsible. "" In addition, the purpose of the present invention is to use the above-mentioned 1C-binding proteins as antigens for the production of polyclonal and/or monoclonal antibodies against these proteins. Antibodies obtained in this way can, in turn, be used to purify new 1C-binding proteins proteins from various sources, such as cell extracts or transformed cell lines. These antibodies can also be used for diagnostic purposes, for example, to identify

Ge) disorders associated with a violation of cellular functions mediated by receptors belonging to the 5th superfamily of TMP/MCE receptors. o Another goal of the present invention is to obtain pharmaceutical compositions containing the aforementioned 1C-binding proteins and pharmaceutical compositions containing an antagonist of 1C-binding proteins for the treatment or prevention of TME-induced /- or

EA5-ligand-induced compound; moreover, the indication of the composition can be used to enhance the action of TME or RAZZ-ligand, or to inhibit the action of TME or RGazZ-ligand, depending on the nature of the above-mentioned 1C-binding protein or its antagonist contained in this composition. iF) In addition, in accordance with one more purpose of the present invention, other methods of elimination or inhibition of endogenously formed or exogenously introduced TME or RAB-K-ligand by using soluble oligomeric TME-receptors, because oligomeric RAZ-receptors, are disclosed herein. or oligomers, which are a mixture of TME receptors and

EAB receptors. In this regard, it should be noted that one attempt was made to isolate and produce, using the recombinant DNA technique, a TME-binding protein called TVR-1, which, as it was shown, had the ability to inhibit the action of TME. The antagonism of the specified protein was determined by measuring the decrease in the cytotoxic activity of TME, as well as by measuring the level of ego b5 Mnhibirovaniya svyazushvania TME with its receptors (EP 308378). As it was discovered, TVR-1 protects cells from the toxic effect of TME at concentrations of several nanograms per milliliter, and prevents the binding of TME-o and TME-B to cells when this protein is simultaneously introduced with cytokines. A subsequent study of the mechanism by which TVR-1 acts revealed that TVR-1 does not interact with the target cell, but most likely blocks the function of TME by specifically binding to TME, competing with its receptor.

Later, using the second purification method, the presence of two active components was discovered, one of which is TVR-1, and the second is the second TME-binding protein, which we named TVR-11 - (first described in (EP 3983271). Both proteins provided protection of cells from the cytocidal and myo-action of TME, and both proteins bound to TME-B less efficiently than to TME-y. Although in the analysis, 70 electrophoresis performed in PAAG with DSN, it was established that proteins TVR-1 and TVR-11 have a very close molecular weight, however, they can be distinguished from each other by the absence of immunological cross-reactivity, by the difference in the M-terminal amino acid sequences, and by the difference in the amino acid composition.

However, the above-mentioned TME-binding proteins are monomeric and capable! bind to only one monomer of the TME homotrimer, the natural ligand, as a result of which this TME still remains active (ie, incompletely neutralized) due to the fact that this trimer has two more active monomers that are not bound by TME-binding proteins. In addition, so far there have been no descriptions of soluble RA5B-K (soluble proteins that bind to the RA5B-K ligand) capable of binding to the ligand for RA5-K, which is known to be a homotrimeric molecule associated with the cell surface.

The so-called "death domain (of cells)" of the p55-TME receptor (designated p55-1C) was described in Irabot

Tapadiia et al. (1993)), however, in that work it was not shown (as it is done in the present application) that rb55-1C and its "death domain" are self-associating, and this self-association is mainly responsible for signal transmission, leading to inducing a cytotoxic effect on the cell. In addition, in this publication, nothing is reported about the possibility of producing soluble oligomeric TME receptors or soluble oligomeric Raz receptors, and it is also silent about other TME-associated effects induced by p55-1Sb or its fragments, for example, such zffects, such as inducing the expression of the 11-8 gene, which are considered in the present invention. In the second paper, published after the submission of this application, the ability to aggregate (i.e., to self-associate) ro55-1C is revealed, but nothing is mentioned either about the production of soluble oligomeric TME receptors and RA5 receptors, or about other TMEs - associated effects induced by the ligand-independent method of p55-1C or its fragments disclosed in this application. Gee!

During the work on the present invention, we discovered new proteins capable of binding to the intracellular domain of the r5-TME receptor (p551C-binding proteins), the p75-TME receptor and (p751C-binding proteins), and the RAb receptor (RAB -1C-binding proteins). Зты р5Б1сС, р7/51С and h-

EA5Z-1C-binding proteins can act as mediators or modulators of the effector action of TME or RGAB-K-ligand on cells by mediating or modulating the intracellular signal transmission that occurs after binding of TME to rbo5b5- and/or p75-TMP-K or binding PHA5-K-ligand on the cell surface. In addition, it was unexpectedly discovered that rob51C and BAB-1C have the ability to self-associate, and what a fragment! p551C and EA5-1C are equally capable! connect with ro5-1C, especially with the so-called "death domains (cell) (00), located in the intracellular domains (1C) of these receptors, i.e., rOBOY and RHA5B-0O. Thus, ro5-1C and RA5B-1C and their fragment! are also proteins capable of binding to ro51cC and EAZ-1C, and therefore, they can act as modulators of the action of TME or EA5-K-ligand on cells.

In addition, in accordance with the present invention, the nature of the binding -I of one of the new proteins (designated here described as protein 55.11) with the intracellular domain of the rob-TME receptor was more fully disclosed (see Example 1).

Moreover, in its second aspect, the present invention is based on the discovered fact that (Ce) the intracellular domain of the p55-TME receptor (p55-1C); the area contained in it (the so-called "domain of death" p55-1C); and also the intracellular domain of the Raz/APO1 receptor (Raz-1C); and the area contained in it

Mothers (the so-called "domain of death" Raz-1C) have the ability to self-associate. Accordingly, it is possible, with the help of standard recombinant DNA techniques, to construct a soluble oligomeric

The TME receptor, which is a hybrid product containing, at one end, at least two extracellular domains of the TME receptor, and at the other end, at least two of the above self-associating intracellular domains or their fragments, where as a result Such self-association results in an oligomer having at least two hybrid products joined together.

By such UBRUS, this soluble oligomeric TME receptor is capable of binding to two monomers of the natural TME homotrimer, and as such effectively neutralizes the activity of TME.

Neutralization of TME activity is desirable in all the above-mentioned conditions, where there is an excessive amount of TME produced xenogeneously or introduced xogenously, leading to undesirable side effects. In addition, the effective binding of TME to soluble oligomeric receptors of the present invention can also serve to bind xogenously added TME and its subsequent desirable prolonged release in conditions where the introduction of TME has a beneficial effect, for example, in tumor therapy. In a similar way, using standard recombinant DNA techniques, an oligomeric EAb receptor can be constructed, which is a hybrid product containing at least two extracellular domains of the Rab3 receptor at one end, and at least two more self-associating domains intracellular domains or ego fragments, where as a result of such self-association, an oligomer is formed, which has at least two hybrid products joined together. Thus, this oligomeric RGAZ-K is capable of binding to two monomers of a natural homotrimer

EAB-K-ligand, and effectively neutralize the activity of EAB-K-ligand. Neutralization of activity

EAB-K-ligand is desirable in all the above-mentioned situations, where an excess amount of this ligand is associated with undesirable side effects. Similarly, if we accept in 7/0 Attention recent messages indicating a possible connection between TME and

EAB-K-ligand-induced effects on cells, and, consequently, on their possible geographical association on the cell surface, where they bind to their receptors, it becomes obvious that with the help of standard recombinant DNA techniques, it is possible to construct a mixed oligomeric receptor with specificity as to TME, as well as to REA5Z-K-ligand. Such a mixed oligomer must be a mixture of the above-mentioned hybrid products containing at least one extracellular domain of the TME receptor and at least one extracellular domain of the RAbZ receptor at one end, and at least two of the above-mentioned self-associating intracellular domains or their fragments at the second end, where as a result of such self-association, a mixed oligomer is formed, having at least two such hybrid products, joining together. Thus, the specified mixed oligomer is capable of binding at the same time to at least one monomer of TME and one monomer of ERAB-K-ligand, thereby contributing to the reduction or effective neutralization of the activity of TME and ERAB-K-ligand of the cell surface in conditions where, as indicated above, excess amounts of these two cytokines are associated with undesirable side effects on the cells. As indicated above, RAZ-K-ligand is mainly associated with the cell surface, and in recent publications there are also descriptions of forms! TME, associated with the cell surface. Therefore, the indication of TME-K/RAB-K mixed oligomer is especially effective for neutralizing i) the activity of TME and PHA5-K-ligand on the cell surface.

Accordingly, the present invention relates to a DNA sequence encoding a protein capable of binding to one or more intracellular domains of one or more sozo receptors belonging to the tumor necrosis factor/nerve tissue growth factor (TNF/NSF) superfamily of receptors. at

In particular, the present invention relates to a DNA sequence selected from the groups that include: a) a cDNA sequence originating from the region coding for a native protein that binds to ise)

Зз5 Intracellular domain of TME-K and i-

B) DNA sequences capable of hybridization with DNA a) under conditions of moderate stiffness, and encoding a biologically active protein that binds to the intracellular domain of TME-K; c) DNA sequences that are degenerates in relation to the DNA sequences defined in a) and B) as a result of the degeneracy of the genetic code, and which encode a biologically active protein that binds to the intracellular domain of TME-K. The present invention also relates to a DNA sequence selected from the group that includes: a) cDNA sequence originating from the region coding for the native protein, which binds to the intracellular domain of GAZ5-K.

B) DNA sequences that are capable of hybridization with cDNA (a) under moderately stringent conditions, and -I that encode a biologically active protein that binds to the intracellular domain of the EAZ receptor; c) DNA sequences, which are considered identical to DNA sequences,

Me. defined in a) and b), as a result of the genetic code's heterogeneity, which is coded biologically

Ge) an active protein that binds to the intracellular domain of EA5-K.

In specific embodiments of its implementation, the present invention relates to DNA sequences encoding proteins that bind to intracellular domain 4) of the β-TME receptor, β-TME receptor, and HA5 receptor, for example, to DNA sequences , encoding proteins, designation 55.1; 55.3; 55.11; 75.3; 75.16; 2; EHR and 0011.

The present invention also relates to proteins, their analogs or derivatives, encoded by any of the above-mentioned sequences of the present invention, and capable of binding to one or more intracellular domains of one or more TME receptors or EAbB receptors. (F) A variant of this aspect of the present invention includes proteins designated 55.1, 55.3, 55.11, 75.3, ka 75.16, 22, E9 and 0011, their analogues and their derivatives.

The present invention also relates to vectors encoding multiple expression of the protein of the present invention and containing multiple expression of the DNA sequence of the present invention; moreover, the indication is a vector! capable of being expressed in the corresponding zukaryotic or prokaryotic host cells. In addition, the present invention relates to transformed zukaryotic or prokaryotic host cells containing the vector!; and to the method of producing proteins, their analogs or derivatives of the present invention by cultivating such transformed b5 host cells in conditions suitable for the expression of the indicated proteins, with subsequent post-translational modification of the indicated proteins, if necessary, and extraction of the expressed protein, analogue or derivative from the culture medium of the specified transformed cells or from cell extracts of the specified transformed cells.

In another aspect, the present invention relates to antibodies or their active derivatives or fragments specific to the proteins, analogues and derivatives of the proteins of the present invention.

In another aspect, the present invention relates to the use of DNA sequences of the present invention or proteins of the present invention encoded by these sequences; moreover, the use provides for the implementation, in particular, of the following methods: the method of modulating the action of TME or RAB-K-ligand on cells carrying the TME receptor or 7/0. EAZ-receptor, consisting in the processing of indicated cells with one or more proteins selected from the group, which includes proteins, analogs and derivatives of the present invention, and proteins rb551C, ro5OO

EAB-1C or EBAB-OO, their analogs or derivatives, where all protein indications are capable! bind to the intracellular domain and modulate the activity of TME-K or EBA5B-K, and the specified treatment of cells provides for the introduction into cells of one or more proteins, their analogs or derivatives in a form suitable for intracellular administration; or the indicated processing involves the introduction into the cells

DNA sequences, in the form of a suitable zkpressing vector, encoding one or more proteins, their analogs or derivatives; i) a method of modulating the action of TME or BABZ-K-ligand on cells carrying TME-K or EAB-K, which consists in treating those cells with antibodies, their active derivatives or fragments of the present invention; ii) a method of modulating the action of TME or RAB-K-ligand on cells carrying TME-K or EAZ-K, which consists in treating those cells with an oligonucleotide sequence encoding a sequence that is antisense, at least to part of the sequence of the present invention; or an oligonucleotide sequence encoding a sequence that is antisense to the sequence of po51C, po5O0O, RA5B-1C or RGAB-0O; moreover, the indicated oligonucleotide sequence is capable of blocking the expression of at least one of the proteins that bind to the intracellular domain of TME-K or GA5-K; iim) a method of modulating the action of TME or TME-K-ligand on cells carrying TME-K or EGAZ5-K, providing: so z a) construction of a recombinant vector originating from an animal virus and carrying a sequence encoding a protein of the viral surface, which capable of binding to a specific receptor on the cell surface, and a sequence selected from an oligonucleotide sequence encoding a sequence that is anti-sense, at least to a part of the sequence of the present invention, and an oligonucleotide sequence encoding

335 is a sequence that is antisense to the sequence of ro51C, ro5rO, gAZ-1C, or RABZ-OO, and when the indicated virus is introduced into the cell, the indicated oligonucleotide sequence is capable of blocking the expression of at least one of the proteins that bind to the intracellular domain TME-K or EA5-K; and

B) infection of the specified cells with the vector specified in a); m) a method of modulating the effect of TME or RBAB-K-ligand on cells carrying TME-K or EAZB-K, which consists in processing these cells with the corresponding vector encoding a ribozyme having a sequence specific to the Kk sequence selected from the mRNA sequence , with a protein encoding analog or derivative of the present invention, and an mRNA sequence encoding po51C, po50O, BAB-1C or RA5-OO; moreover, the specified sequence of ribozymes! is capable of interacting with an mRNA sequence, and such is capable of cleaving the indicated -I mRNA sequence, which leads to inhibition of expression of a protein, analogue or derivative of the present invention or to inhibition of expression of rb551cS, ro5rO, RHA5B-1C or RA5B-OO;

Me, mi) method of treatment of tumor cells or HIV-infected cells or cells susceptible to a second disease, providing for: a) construction of a recombinant vector originating from an animal virus and carrying a sequence encoding a protein of the viral surface, which has the ability to bind to this a receptor on the surface of a tumor cell or a receptor on the surface of an HIV-infected cell, or which has the ability to bind to a second receptor on the surface of a cell subject to a second disorder; and the sequence selected from the sequence of the present invention, which encodes a protein, analog or derivative of the present invention, and the sequence that co-iodrates roBb1cS, ro5ro,

EAB-1C, RAB-0O, or their biologically active analog or derivative; moreover, the indicated protein, analogue or (F) derivative of the present invention, or ro51cS, ro50O, RHA5B-1C, RHA5B-OO or their analogue or derivative, when introduced into the indication tumor or HIV-infected cells or cells with other disorders, possess the ability to destroy those cells; in 5) infection of the indicated tumor cells or HIV-infected cells or cells with other disorders with the vector defined in (a); miii) method of isolation and identification of proteins, factors or receptors capable of binding to the proteins of the present invention, binding to the intracellular domain, where the specified method provides for the implementation of affinity chromatography, in which the specified protein of the present invention is bound to a matrix for affinity chromatography, and this binding protein put in contact with a cell extract, after which proteins, factors or receptors from that cell extract,

which bind to the indicated binding protein, are separated, isolated and analyzed; ii) of the method of isolation and identification of proteins capable of binding to the proteins of the present invention that bind to the intracellular domain, where the specified method provides for the preparation of a yeast two-hybrid system, in which the sequence encoding the specified protein binding to the intracellular domain is carried by one hybrid vector, and the sequence , originating from a cDNA or genomic DNA library, carries a second hybrid vector, after which the vector used for transformation of host yeast cells is whitened with subsequent extraction of the second hybrid vector to obtain a sequence encoding a protein that binds to the specified 7/0 binding protein with an intracellular domain; and their) method of isolation and identification of a protein capable of binding to intracellular domains

TME-receptors or RHA53-receptors, providing for the implementation of Southern hybridization under non-stringent conditions followed by RSC cloning, in which the sequence of the present invention or its part is used as a probe for hybridization with sequences from the cDNA library or genomic DNA library, ) having at least partial homology with the indicated sequences, and then the indicated hybridized sequence is subjected to amplification and cloning with the help of the polymerase chain reaction (PCR), resulting in a clone encoding proteins possessing at least partial by homology to Kk instructions to the sequences of the present invention.

The present invention also relates to a pharmaceutical composition for modulating the action of TME or

EAb-ligand for cells containing as an active ingredient any one of the following components: i) the protein of the present invention, or such a protein as p51cS, p5OO, RA5B-1C or RA5B-OO, its biologically active fragment), analogs, derivatives or their mixtures; (HER) a recombinant vector originating from an animal virus and encoding a viral surface protein capable of binding to TME-C or GAZ-K-0O receptors of carrier cells or tumor cells, and a sequence that encodes a protein, analogue or derivative of the present invention , or a sequence that leads to ro51cS, ro5ro, and)

EAB-1S or RGAZ-OO; (ii) a recombinant vector originating from an animal virus and encoding a viral surface protein, defined above, and an oligonucleotide sequence encoding a sequence that is antisense to the sequence rob51cS, ro5OO, gAB-1C with zo Mly RAZ-OO; and im) a vector encoding a ribozyme with a sequence capable of interacting with

an mRNA sequence encoding a protein, analog or derivative of the present invention, or with and,

mRNA sequence encoding p551C, p5rO, RAB-1C or EAZ-0O. Gee!

In a specific embodiment of the above aspects, the present invention relates to the use of p55-1C or DNA encoding p55-1C. This variant of the invention is based on the discovery of the fact that (iso) ro5-1C can induce other TME-associated effects in cells in a ligand (TME)-independent manner. Accordingly, the present invention relates to the induction method

TME-associated effects in cells or tissues, which provides for the treatment of indicated cells with one or more proteins, their analogs or derivatives, where the indicated one or several proteins are selected from proteins, all of which are self-associating intracellular domains of ro5-TME-K " (ro5-1C ) or their parts capable of self-association and inducing the TME effect in cells of ligands with a (TME)-independent image; moreover, the indicated processing of cells provides for the introduction of cells into the cells

And the indications of one or more proteins, their analogs or derivatives in a form suitable for intracellular introduction, or the introduction into the cell of a DNA sequence encoding the indication of one or more proteins, their analogs or derivatives, in the form of a suitable vector carrying the sequence, and able to enter this sequence into the cells so that! zta -I sequence could be expressed in zth cells.

Variants of the above-mentioned method of the present invention include:

Me. th method, where the indicated treatment of cells is carried out by transfection of the indicated cells with a recombinant vector based on an animal virus; moreover, the indicated method includes the following stages: o a) construction of a recombinant viral vector carrying the sequence encoding this viral surface protein (ligand) capable of binding to a specific receptor on the surface of the indicated treated cells; and the second sequence, which controls the p55-1C protein or its fragment, analogs and derivatives, where the indicated protein, when expressed in the indicated cells, is capable of self-association and induction of one or more TME-associated effects; and 5) infection of the specified cells with vector (a);

F) and) the method, where the specified TME effect induced in the specified cells is the induction of the expression of the 11-8 gene, and the specified vector is a vector carrying the sequence encoding, basically, the entire specified p55-1C, its fragment, analogues and the derivative, which, when they are expressed in the cells, is capable! to self-association and signal transmission for inducing the expression of the specified gene 11--8; her) a method of treating tumor or virus-infected cells, or a method of enhancing the antibacterial action of granulocytes, where the indicated viral vector carries a sequence encoding a viral ligand capable of binding to a specific receptor on the surface of the indicated tumor cells, virus-infected cells or granulocytes, and the sequence, 65 Encoding the expression ro5-1C, its fragment, analogs and derivatives, which when expressed in the indicated tumor cells, virus-infected cells or granulocytes, induce a TME-associated effect, leading to the death of those cells; them) a method of treatment of tumor cells, where the indicated ro5-1C, its fragments, analogs or derivatives, when expressed in tumor cells, induce the expression of 11/-8, which induces cytolysis of the indicated tumor cells due to their chemotactic activity, which attracts granulocytes and other lymphocytes to tumor cells, and leading to the death of tumor cells.

In this aspect, the present invention also relates to the intracellular domain of p55-K, (p5-1C) its fragments, analogs and derivatives, used to treat cells by inducing TMP-associated effects in these cells; and to the following ego variants: 70 m) rob5-1C, ego parts, analogs, and derivatives for use in processing cells by inducing the expression of the 11-8 gene in them. mi) ro5-1C, its parts, analogues and derivatives for use in the treatment of tumor cells by inducing in them the expression of the 11-8 gene, which leads to the death of those tumor cells.

In addition, in this aspect, the present invention relates to a pharmaceutical composition for the treatment of cells by inducing TME-associated effects in them, where the specified composition contains p5B-1C, its fragment, analogs, and all their derivatives as an active ingredient, and a pharmaceutically acceptable carrier; as well as the following variants of this pharmaceutical composition: i) a pharmaceutical composition for treating cells by inducing TME-associated effects in them, containing as an active ingredient a recombinant vector originating from the Animal virus and encoding p55-1C, its fragment, analogs, and all their derivatives, and protein capable of binding to the surface protein on the processed cells; i) a pharmaceutical composition for the treatment of tumor cells, the introduction of which leads to the induction of 11-8 expression, and the subsequent death of tumor cells.

In its second aspect, the present invention relates to a soluble oligomeric receptor for tumor necrosis factor (TME-K), consisting of at least two self-associating hybrid proteins, each of which has a) at one end, a TME-binding domain , selected from i) the extracellular domain of TME-K, its analogs or derivatives; and the specified extracellular domain, its analogs or derivatives are incapable of unwanted self-association, and capable! communicate with TME; and c) at its other end, the self-associating domain, selected from (i) the mostly complete intracellular domain of roo-TME (ro5-1C), extending approximately from amino acid residue 206 to approximately amino acid residue 426 of the native molecule! p55-TME receptor (p55-K); ii) "death domain" p5B5-1C, extending approximately from amino acid residue 328 to approximately amino acid residue 426 b of native p55-K; (ii) mainly, the complete intracellular domain of the RGAB/AROT1 receptor (EA5B-1C); i) "domain of death" RAB-1S; and m) analogs, fragments, or derivatives of any of (1I)-(im), possessing the ability to self-associate) with 5 self-association, where at least two self-associating proteins undergo self-association only at the indicated ends (B), and in svojy vykonos (a), zty squirrels are capable! bind to at least two TME monomers, and each of the ends (a) is capable of binding to one TME monomer; and in addition, the present invention relates to salts and functional derivatives of the indicated soluble oligomeric TME-K. "

Option! This aspect of the present invention includes all combinations of the multiple ends in c (a) with ends (b), so, for example, these variants include soluble oligomeric TME-K, containing as an extracellular domain the extracellular domain of rob5-K, and as self-associating;" the p5o5-16 domain of the intracellular domain.

In addition, the present invention relates to a method of obtaining soluble oligomeric TME-K, providing: - and a) construction of an expression vector encoding any one of the indicated hybrid proteins, where the DNA sequence of each of the indicated ends of the hybrid protein is obtained from cloned me) DNA- sequences encoding mainly the complete extracellular domain of TME-K, its analogs or

Ge) production; and from the cloned DNA sequences encoding, mainly, the complete ro5-1C, the "5o death domain" ro5-1C, Rav-1C, the "death domain" Rav-1C, and all their analogues and derivatives; and the end! they are ligated to each other, forming a sequence of a hybrid protein, which is then inserted into the specified vector under the control of the sequences that regulate transcription and translation;

B) introduction of the vector (a) into the corresponding host cell, in which the specified hybrid protein is expressed; and 5B c) purification of the hybrid protein expressed in the specified host cells; moreover, the specified hybrid protein is subject to self-association before, during, or after the cleaning process, as a result

F) by which soluble oligomeric TME-K is obtained. In addition, the present invention relates to a vector encoding a hybrid protein used in the above-described method of the present invention; to host cells containing the vector; and a soluble pharmaceutical composition, which includes soluble oligomeric TME-K, its salts or functional derivatives and their mixtures as an active ingredient in combination with a pharmaceutically acceptable carrier. Similarly, in accordance with the present invention, soluble oligomeric TME-K, its salts, functional derivatives and any of their mixtures can be used to inhibit the undesirable action of TME in mammalian cells, and in particular, for the treatment of complex conditions caused by the excess presence of endogenously formed or xogenously introduced TME; or, alternatively, they can be used for long-term maintenance of the beneficial effect of TME in mammalian cells with xogenously introduced TME.

In accordance with the above-described aspect of the present invention, it should also be noted that it is possible to construct a soluble oligomeric EAB/AROT1 receptor (GAZ-K), which can be used to inhibit the unwanted action of the PHA5Z-ligand. Therefore, in another aspect, the present invention relates to the soluble oligomeric EAB/APO1 receptor (EGAZ-K), consisting of at least two self-associating hybrid proteins, each of which has (a) at one end, a domain, a bond binding with the RAZZ-ligand selected from the extracellular domain of BAZ5-K, its analogs with derivatives incapable of self-association and capable of binding to the RAZZ-ligand; and B) at its second end, the 70 self-associating domain, selected from (i)) mainly, the complete intracellular domain of RB5B-TME-K, (p55-1C), extending approximately from amino acid residue 206 to approximately amino acid residue 426 of the native molecule p5 -TME-K (p5B5-K); (ii) "death domain" rb55-1C, extending from approximately amino acid residue 328 to approximately amino acid residue 426 of native p5o5-K; (her) mainly, the complete intracellular domain of the RGAB/APO1 receptor (RGAZ-1C); (imu) "domain of death" 7/5 RAZ-1C; and (M) analogs and derivatives of any of (iI)-(m), possessing the ability to self-associate, where at least two self-associating proteins undergo self-association only at the indicated ends (B), and have the indicated ends (a), capable of binding to at least two monomers of the EAbZ-ligand, and each of the ends (a) is capable of binding to one monomer

EAb-ligand; and in addition, the present invention relates to salts and functional derivatives of the specified soluble oligomeric GAS receptor.

In accordance with this aspect, the present invention also relates to a method of obtaining soluble oligomeric GAB-K, which provides for: a) construction of an expression vector encoding one of the two specified hybrid proteins, where the DNA sequence of each of the specified ends of the hybrid protein is obtained from cloned

DNA sequences encoding, mainly, the complete extracellular domain of BAZ-K, its analogs or derivatives; and from the cloned DNA sequences encoding, basically, the entire ro5-1C, "domain (8) of death" ro5-1C, EA5B-1C, "death domain" GA5-1C, all their analogues and derivatives; and the end! ligate together, forming a sequence of a hybrid protein, which is then inserted into the specified vector under the control of transcriptional and translational regulatory sequences; b) introduction of vector (a) into the corresponding host cell, in which the specified hybrid protein is expressed; and c) purification of the hybrid protein expressed in the indicated host cells, and the indicated hybrid protein is subjected to self-association before, during, or after the purification process, resulting in soluble oligomeric EA5-K. ise)

In addition, the present invention relates to an expression vector containing a sequence encoding a soluble oligomeric RA5B-K used in the above-described method; to host cells containing the specified vector; and to pharmaceutical compositions that include soluble oligomeric RAZ-K, its salts or functional derivatives, or any mixture thereof as an active ingredient in combination with pharmaceutically acceptable carriers. Similarly, the present invention relates to the soluble oligomeric GAZ-K, its salts, or functional derivatives or any of their 7) mixtures, which can be used to inhibit the undesirable action of the ГА5Z-ligand in mammalian cells, and in particular, for the treatment of due to their excessive presence, nodogenically;" formed or xogenously introduced GAZ-ligand.

Analogously to the above description, which concerns TME oligomeric receptors and RAB oligomeric receptors, it is also possible to construct a mixture of oligomers capable of specifically binding to both TME and RA5-K-ligand. Thus, the present invention also relates to mixed TME-K/RA5B-K oligomers consisting of at least two self-associating hybrid proteins,

Me. one of which is selected from any of the above TME-specific hybrid proteins, and the other is selected from any of the above GAB-K ligand-specific hybrid proteins, resulting in a mixed oligomer having at least one TME-K extracellular domain, and, at least, one about the extracellular domain of EBAB-K, the connection together due to self-association between these intracellular domains or their fragments, hybridizations with each of these extracellular domains. You are a mixed-up oil-homer receptor! constructed by obtaining, as described above, oligomeric TME receptors and oligomeric RGAb receptors, their subsequent mixing, and then selecting, by standard methods, those oligomers capable of specific binding to BABZ-K-ligand and TME. Another method of obtaining mixed oligomeric receptors is co-transfection of suitable host cells

F) vectors (described above) encoding any of the TME-specific hybrid proteins (soluble and TME receptors) and encoding any of the EAB-K-ligand-specific hybrid proteins (soluble

EAB receptors); purification of expressed hybrid proteins, which undergo self-association before, during, and after purification, as a result of which oligomeric receptors are obtained; and the subsequent selection using a standard technique of those oligomeric receptors that have the ability to bind to

TME and GAB-K-ligands.

Similarly, it is possible to prepare pharmaceutical compositions containing a mixture of oligomeric receptors, their salts or functional derivatives, or any of their mixtures as an active ingredient in combination with a pharmaceutically acceptable carrier. In addition, the present invention relates to mixed oligomeric receptors, their salts or functional derivatives or any of their mixtures, which can be used! for the inhibition of the undesirable effect of TME and HA5-K-ligand in the body of mammals, and in particular, for the treatment of conditions caused by the excessive presence of xenogeneously formed or xogenously introduced TME and RGAZ-K-ligand; or alternatively, they can be used for long-term (prolonged) maintenance of the beneficial effect of TIME and/or

EAB-K-ligand in mammalian tissues when TME and/or RHA5-K-ligand (in soluble form) was introduced xenogeneously.

Other aspects and variations of the present invention will also be apparent from the following detailed description of the invention. 70 It should be noted, however, that the terms "modulation of TME action on cells" and "modulation of RAZ-ligand action on cells" are used in the present description to mean the treatment of both miggo and mimo.

Figure 1a-4 schematically shows an incomplete and preliminary nucleotide sequence

cDNA clones encoding ro5bB1C- and p751C-binding proteins, and the derived amino acid sequence of protein 55.11, where Fig. 1(a) shows the sequence (ZEO IU MO:9) of clone 55.11, 7/5 Encoding rob51C-binding protein 55.11; in Fig. 1(5) shows the partial and preliminary sequence (ZESO IU MO:10 and BEO IU MO:11) of clone 75.3, which controls p751C-binding protein 75.3; and Fig. 1(c) shows the partial and preliminary sequence (ZEO IYU MO:12 and ZEO IYUO MO:13) of clone 75.16, which encodes p751C-binding protein p75.16 (all indications of the sequence of descriptions! in

Example 1); and Fig. 1(4) shows the deduced amino acid sequence of protein 55.11 (ZEO IU

MO: 14), obtained on the basis of the nucleotide sequence shown in Fig. 1(a), and described in

Example 1.

Fig. 2 illustrates the Northern blot analysis, which revealed the presence of 55.11-specific mRNA in a number of tested cell lines, as described in Example 1.

Figs. ZA and ЗV show autoradiograms illustrating the binding of a protein encoded by two 29.11 cDNAs to o5T-hybrid proteins containing parts of p5b5-1C; where Fig. ZA illustrates the binding of the full-size protein 55.11 (55.11-10I) with various О5T-hybrid proteins; and Fig. 3B i) illustrates the binding of the 55.11 part linked to the RI AC octapeptide with various NWT hybrid proteins (all of them are described in Example 1).

Fig. 4 schematically illustrates the comparison of the deduced amino acid sequence of human 55.11 (ZEO IU MO:14) with the sequences of related proteins originating from lower organisms:

UNKO27s (yeast; ZEO IYUO MO:15), BEMZ (yeast; ZEO IYUO MO:16), A. (Naiiyapa (plant; 560 IYU MO:17) and S. o eiedapz (nematode; ZEO IYU MO:18), as presented in Example 1. Gee!

Figure 5 shows a Western blot stained with a polyclonal antiserum against MVR and illustrating the self-association of roB1C, where the Western blot was obtained from a SDS-PAGE gel on which electrophoresis of interacting bacterially produced chimeric proteins ro51C-MVR and yrob510 was performed. 517 (lanes 1-4) or the control (interaction between the chimeric protein ro51C-MVR and one OT (lanes 5-8); however, the interaction between the chimeric proteins (and the control) was performed on glutathione-agarose beads before electrophoresis on PAGE with SDS , as described in Example 2.

Figure 6 shows! phase-contrast photomicrographs illustrating the cytotoxic effect of "full-size ro51C in Na cells) transfected with an expression vector encoding the protein Che) with roB51C (right panel); and inhibition of that cytotoxic action in the event that vector expression is blocked by treatment of cells with tetracycline (left panel) as described in Example 2. Fig. 7 illustrates the ligand-independent stimulation of the cytocidal effect in Neg cells transfected with full-length p55-K, its intracellular domain, or parts of the intracellular domain, including the "death domain", where: different DNA molecules encoding full-length P5BB5-VM, its intracellular domain and fragment! of the intracellular domain, which was

Me. insert into the vector with which Her cells were transfected! and.

Ge) ii) left and middle column! the graph illustrates the expression of the TME receptor in Her cells and for each

Of the receptor types shown in the very left part of Fig. 7, the left column represents the amount of the receptor in ng/cell. sample, and the middle column of the graph represents the amount of the receptor, expressed in these units of radioiodinated TME, associated with transfected cells; and ii) the right column illustrates the variability of Neia cells expressing different types of receptors; and where: in all graphs, the unshaded rectangles represent cells transfected in the presence of tetracycline, and the shaded rectangles represent cells transfected in the absence of tetracycline (all indications are described in Example 2).

F) Figure 8 illustrates the ligand-independent induction of 11/-8 gene expression in Nei cells transfected with full-length rob5-K or its intracellular domain (ro551C), where Northern blot analysis of RNA is illustrated in panel A. extracted from Neia cells treated or in untreated TME (two left lanes, labeled "control" and "TME"), and RNA extracted from NeHa cells transfected with vectors encoding p55-K, p55-1C or the control protein, luciferase ( the rest of the track, marked "p55-1C", "p55-K", and "is", respectively); however, in each case, cells were transfected in the presence (Ж) or in the absence (-) of tetracycline (therefore, there are two lanes for transfected cells); And where in panel B is illustrated the staining of 185 65 rRNA with methylene pigeon in each of the samples of Her cells! and, shown in panel A (all the above-mentioned options are described in Example 2).

Fig. 9 (A and B) graphically illustrates the ligand-independent stimulation of the cytocidal effect in the cells of Ne! a, transfected ro5K or its fragments, or GAB-1C, where in Fig. 9A the results for roK or their fragments are presented, and in fig. 98 views! results for GA5-1C. On the left, in panels A and B, are schematic images of parts of rob5K or EA5Z-1C, used for transfection, and on the right in those panels are graphical representations! experimental result! (all indications are options! descriptions! in

Example 2).

Figure 10 schematically shows a partial and preliminary nucleotide sequence (5EO

IO MO:19 and ZEO IU MO:20) cDNA clone (designated "E2"), which encodes a protein capable of binding to 7/0. B951C and GA5-1C, as described in Example 3.

Figure 11 schematically shows a partial and preliminary nucleotide sequence (5EO

IUO MO:21-23) cDNA clone (designated E9), which encodes a protein capable of binding to Rob1isC

EAZ-1C, as described in Example 3.

Figure 12 schematically shows the partial and preliminary nucleotide sequence (5EO 75.10 MO:24) of the cDNA clone (designated ХО11), which encodes a protein capable of binding to РОБ1СС, and in particular РО5ОРО and ГА5-1С, as described in Example 3.

In one of its aspects, the present invention relates to novel proteins that have the ability to bind to the intracellular domain of receptors belonging to the superfamily

TME/MOR, such as the TME receptor (TME-K) and the RAB receptor (RGAB-K), and which, therefore, are recognized as mediators or modulators of that superfamily of receptors, for example, TME-K and EAZB-K, playing a certain a role, for example, in signal transmission initiated by binding TME to a receptor

TME and RABZ-ligand with the RAB receptor. Examples include proteins that bind to the intracellular domain of p75-TME-K (p551cS), such as the proteins described herein in 55.1, 55.3, and 55.11 (Example 1), as well as proteins encoded by E2 cDNA clones. BU and 0011 (Example 3); proteins that bind to the intracellular domain of p/5-TME-K (p/51C), such as proteins designated 75.3 and 75.16 (Example 1); and proteins that bind to the intracellular domain of BAB-K (BAZ-1C), such as proteins, and) encoded by cDNA clones E2, E9U and 0011 (Example 3). It was established that proteins 55.1 and 55.3 represent parts or a fragment! extracellular domain p55-TME-K, (rb551C); and other proteins, namely, 55.11, 75.3 and 75.16, were not described at all before the present invention (75.3, 75.16), or they were described (55.11, see, with Kpap et al., 1992), but about their functions or other properties, in particular, nothing is reported about the ability to bind to TME-K (see below, Example 1). The new proteins encoded by cDNA clones E2, BU and 0011 also represent proteins that have not been previously described, i.e., their sequences are not available in DNA data banks ("ZEMEVAMK") or in amino acid sequence data banks ("RKOTEI!M VAMK").

Thus, the present invention relates to DNA sequences encoding protein instructions and to proteins encoded by these DNA sequences. Cha

In addition, the present invention also relates to DNA sequences encoding biologically active; analogues and derivatives of these proteins, and to analogues and derivatives encoded by these sequences. The indicated analogs and their derivatives are obtained by standard methods (see, for example, ZatrbgookK et al., 1989), where, in the DNA sequences encoding these proteins, one or more codons may be deleted, added, or replaced, so that! the resulting analog had a change in at least one amino acid compared to the native protein.

Acceptable analogs are those analogs that retain, at least, their ability to;" binding to the intracellular domain of a receptor belonging to the TME/MOBE receptor superfamily, such as REAB-K, or TME-K, for example, p51C, p751C, or RA5B-1C, or which can mediate any other binding or enzymatic activity, for example, such analogs that bind to robo, -I p751C or RAB-1C, but which do not transmit a signal, i.e., do not bind to a second downstream receptor, protein, or second factor, or do not catalyze a signal-dependent reaction. Thus,

Me. there may be production analogs that have the so-called dominant negative effect, namely, analogs that are defective either in connection relations, for example, with 5o ВРОо1С, р751С or RAZ-1С, or in relations of subsequent transmission of the signal after connection. These analogs can be used, for example, to inhibit the action of TME or RA5-ligand by competing with these natural 1C-binding proteins. In the same way, they can beat productions! so-called dominant-positive analogs that have the ability to enhance the effect, for example, TME or

EAb-ligand. These analogues should have similar or even better 1C-binding properties, as well as similar or even better ability to transmit a signal than natural 1C-binding proteins.

Similarly, derivatives can be obtained by standard modifications of side groups (F, one or more amino acid residues of proteins, or by conjugation of proteins with another molecule, for example, with an antibody, enzyme, receptor and other molecules well known to specialists.

Newer proteins that bind to the intracellular domain of TME-K and BAZ-K, for example, proteins 55.1, 55.3, in 55.11, 75.3, 75.16, as well as proteins encoded by cDNA clones E2, 9 and 0011 (hereinafter referred to as E2, E9 and 0О11 ) can be used for various purposes, for example: i) They can be used to imitate or enhance the function of TME or RHA5B-K-ligand in those cases when enhanced action of TME or EBAB-K is desirable, for example, in antitumor, anti-inflammatory or anti-HIV therapy, where it is necessary to achieve TME- or 65 / EAZ-K-ligand-induced cytotoxicity. In these cases, proteins, for example, proteins that bind to rob1c, such as 5.1, 55.3, as well as E2, E9 and 0011, and directly ro51cS itself (see below, and Example 2), as well as the "death domain" rb551C (p55O0), which enhances the effect of TME; or proteins E2, E9U and 0011, as well as

EAB-1C and RAB-BO, which enhance the effect of RGAZ-K-ligand, i.e., have a cytotoxic effect, can prevent introduction into the cell using a standard technique known as reg ze. So, for example, because

ZI proteins are considered intracellular, and it is desirable that they be injected! only in those cells where it is preferable to induce the action of TME or RA5-K-ligand, then for the specific introduction of those proteins into the cells it is necessary to use a certain system. One of the ways to construct such a system! is the creation of a recombinant animal virus, for example, a virus derived from the cowpox virus, to obtain DNA from which the bats can be taken! and successive introductions! two genes: gene, 7/0 Coding ligand that binds to cell surface proteins expressed by cells, for example, such as the dr12O protein of the AIDS virus (HIV), which specifically binds to some cells (CO4 lymphocytes and related leukemia cells ) or a gene encoding a second ligand that specifically binds to cells carrying TME-K or EAZB-K, so that the recombinant viral vector is capable of binding to cells carrying TME-K or RAZ-K; and the gene encoding a new protein that binds to the intracellular domain, or protein po51C, po5O0O, BAB-1C or RA5B-0O. Thus, a protein on the surface of the virus that binds to the cell surface will specifically direct that virus to a tumor cell or to another cell carrying TME-K or RHA5-K, after which the sequence encoding the protein that binds to the intracellular domain, or the sequence , encoding rbo551cS, ro50O, RAB-1C or RHA5B-OO, will be introduced into the cells with the help of this virus, and after its expression in these cells, an increase in the action of TME will be induced or

EAB-K-ligand, which will lead to the death of tumor cells, or other cells carrying TME-K or EAZB-K, the destruction of which would be desirable. The construction of such a recombinant animal virus can be carried out using standard techniques (see, for example, Zatrgook et al., 1989). The second possibility of introduction of specified proteins into cells can be implemented by introduction of new proteins or p5b51C, p5O0O, BAB-1C or GAB-OO in the form of oligonucleotides, which can prevent absorption! cells and expression in them. i) i) They can be used to inhibit the action of TME or PHA5-K-ligand, for example, in cases of tissue destruction as a result of septic shock, "transplant versus host" type rejection, or acute hepatitis, where it is necessary to block TME- induced intracellular signal transmission (U

TME-K or RA5-K-ligand induced intracellular transmission of the GAZ-K signal. In this case, it is possible, for example, to introduce into the cells, using standard techniques, oligonucleotides with antisense and coding sequences for silent new proteins, or antisense coding sequences (33 for p5B1C, p55OO, BAB-1C or BAB-YOO, which could effectively block the translation of mRNAs encoding these proteins, and thereby block their expression, which, ultimately, would lead to the inhibition of the action of TME or GAZ-K-ligand. uh-

Such an oligonucleotide! can enter cells using the above-described virus; moreover, the second sequence contained in this virus should be an oligo-nucleotide sequence. The second method is the use of antibodies against the indicated proteins, which will lead to inhibition of their ability to intracellular signal transmission. It is possible that these new proteins have an extracellular domain and an intracellular domain, while the intracellular domain binds to TME-K (8) or RGAB-K-binding domain, and then antibodies generated against their extracellular domains can be used ! for blocking TME or GA5Z-K-associated functions. Another method of inhibiting the action of TME or RA5B-K-ligand is a recently developed method using ribozymes. Let's go fishing! are catalytic molecules! RNA that cleaves RNA specifically (ie, has the property of autocatalysis). Ribozymes can be constructed to cleave the necessary RNA, for example, mRNA encoding the new proteins of the present invention, or mRNA encoding p551C, p55O0O, RA5B-1C or RGAB-0O. Such fish winter! duty! to have

Me. a sequence specific for the selected mRNA, as well as must possess the ability to interact with it (complementary binding) with subsequent cleavage of that mRNA, which should ultimately lead to a decrease (or complete suppression) of protein expression, which must be inhibited, moreover, the level of reduction of expression depends on the level of expression of ribozymes! in the target cell. 4) To introduce ribozymes into vibrating cells (for example, into cells carrying TME-K or BA5B-K), any suitable vector can be used, for example, a plasmid, a viral vector! animals (retroviruses!), and vectors that are commonly used for these purposes (see above, where the virus has, as the second in the sequence, a cDNA encoding a selected ribozyme sequence). In addition, ribozymes can be constructed that have multiple targets (multi-target ribozyme!), which can be (F, used, for example, to inhibit the expression of one or more proteins of the present invention and/or ro551C, ro50O, RA5B-1C or EGABZ- OO, review of methods, etc., devoted to ribozymes, see

SNep eyi ai., 1992; 2pao 8. RiskK, 1993; Zpoge ei ai., 1993; dozerp 5 Vigke, 1993; Zpitauata eyi ai., 1993; Sapiog eyi vo a!|., 1993; Vahipada, 1993; Tracks! ei ai!., 1993; Koigiti eyi ai., 1993). her) They can be used! for the isolation, identification and cloning of other proteins capable of binding to them, for example, other proteins involved in signal transmission, which are downstream of the TME-K or BAB-K intracellular domain. In this case, the indication of the variant, namely

DNA sequences encoding these proteins can be used in the yeast two-hybrid 65 system (see Example 1), in which the sequence of these proteins can be used as a "bait" for isolation, cloning and identification from cDNA libraries or genomic DNA of other sequences ("dobies"), encoding proteins that can associate with these new proteins that associate with the intracellular domains of TME-K or RA5B-K. In a similar way, it can also beat concretely. proteins of the present invention, namely, proteins that bind to rob1C, p75C or BAB-1C, bind to other receptors of the TME/MOB receptor superfamily.

For example, it was recently reported by IZspmai et al., 1993; Voepz et al., 1993, Stgome et al., 1994), that in addition to p55- and p75-TME-K, there are other receptors! TME. Accordingly, using the yeast two-hybrid system, it is possible to accurately verify whether the proteins of the present invention have the ability to specifically bind to these second TME receptors or other receptors of the TME/MOP superfamily. In addition, this method can be used to determine whether the proteins of the present invention have the ability to bind to other known receptors, in the activity of which they can play a functional role. them) Zty new squirrels can also be used! for the isolation, identification and cloning of other proteins of the same class, i.e., proteins that bind to the intracellular domains of TME-K or BA5-K 7/5 ml with functionally related receptors, and participate in intracellular signal transmission. In this case, the above-described yeast two-hybrid system can be used, or the recently developed (UMIiK5 et al. 1989) system can be used using a non-strict

Southern hybridization with subsequent RSC cloning. in the publication of UUik5 et al., the identification and cloning of two putative protein-tyrosine kinases using non-stringent 2o Southern hybridization with subsequent cloning by means of PCR based on the known sequence of the "motif" of the putative kinase sequence is described. In accordance with the present invention, this method can be applied using the sequences of new proteins for the identification and cloning of proteins related to proteins that bind to the intracellular domain of TME-K, RA5B-K or a related receptor (receptors of the TME/MOB superfamily). sch m) In another variant, new proteins of the present invention can be used! in the methods of affinity chromatography for the isolation and identification of other proteins or factors with which they i) are capable! bind, for example, to other receptors related to TME-K (receptors of the superfamily

TME/MOP) or other proteins or factors involved in the signal transmission process. In this case, the proteins of the present invention can be separately bound to the matrix used for affinity chromatography, and then exposed to cell extracts or secreted proteins or factors suspected of being involved in the process of intracellular signal transmission. After carrying out affinity chromatography, other proteins or factors that bind to the new proteins of the present invention can be clarified, isolated and characterized. mi) As indicated above, the new proteins of the present invention can also be used as ise) with 5 Mmmunogens (antigens) for the production of antibodies against them. These antibodies can also be used to purify new proteins either from cell extracts or from transformed cell lines that produce them. In addition, these antibodies can also be used! for diagnostic purposes of identification of disorders associated with abnormal functioning of systems

TME or PGA5-K-ligand, for example, cellular effects induced by overactive or underactive

TME or RAB-K-pigands. So, for example, in the case when such disorders are connected! with insufficient functioning of systems! transmission of an intracellular signal, in which new proteins are involved, the indication of antibodies can serve as an important diagnostic tool. ;" However, it should be noted that isolation, identification and characterization of the new proteins of the present invention can be carried out using well-known standard screening methods. So, for example, one of these screening methods (method with obtaining a double yeast hybrid, as described below in Examples 1 and 3) was used to identify new proteins of the present invention. As described above and below, other well-known methods can be used, such as affine

Me. chromatography, DNA hybridization, etc. for the isolation, identification and characterization of new proteins of the present invention, or for the isolation, identification and characterization of other proteins, factors, receptors, etc., which have the ability to bind to the new proteins of the present invention or to receptors belonging to the TME receptor family/ MOR. As for the above-mentioned antibodies, the term "antibody" used herein means polyclonal antibodies, monoclonal antibodies (tAbs), chimeric antibodies, anti-idiotypic antibodies (anti-14), against antibodies that can be labeled in soluble or in a bound form, and soluble in their fragments obtained using known techniques, such as enzymatic cleavage, peptide synthesis or recombinant DNA techniques, etc. (F, Polyclonal antibodies represent a heterogeneous population of antibody molecules obtained from the serum of animals immunized with an antigen. Monoclonal antibodies represent, basically, a homogeneous population of antigen-specific antibodies, which have, basically, the same epitope-binding site. Monoclonal antibodies can is obtained by known methods. See, for example, Kopieg 5 Mivivip, Maishge, 256: 495-497 (1975); US patent Mo4 376 110;

I ape AMTIVOOIE5, A I AVOKATOKU MAMOAIG, Soia Zrgipud Nagbog I arogaiyyugu (1988); and SoiPdap ei ai., eav., Sitepi

Rgoiosoiiv ip Ittypoiodu, Sgeepe ribiiizpipud Avzos. 5 UMPeu Ipiegesiepse M. U., (1992, 1993)3), moreover, the content of those works is introduced into the present description by means of a link. These antibodies can kill as many as 65 immunoglobulins! classes go, 19M, 19E, T9A, ZY O, and their beloved subclasses. Hybridomas producing monoclonal antibodies of the present invention can be used for the cultivation of ip myo, ip 8, and ip mimo.

The possibility of producing a high titer of monoclonal antibodies IP migo or IP zysh makes this method especially preferable.

Chimeric antibodies are molecules consisting of different parts originating from different species of animals, for example, such molecules that have a variable region originating from IgG antibodies and a constant region originating from human immunoglobulin. Chimeric antibodies are used, mainly, to reduce immunogenicity and to increase output during production, for example, such chimeric human/muscle TABs can be used in those cases when muscle TABs have a higher output! from a hybrid, but have increased immunogenicity for humans. Chimeric antibodies and methods of their production are known and described in the literature. May). Asai

All together. PERSON 81: 3273-3277 (1984); Moittizop ei aiYi., Rhos. May Asad si. BOTH 81: 6851-6855 (1984); Voshiappe et al., Maishge 312: 643-646 (1984); Sariyu ei ai,, application for European patent 125023 (published on November 14, 1984); Meiregdeg ei aiYi., Mayte 314: 268-270 (1985); Moitisop ei ai. Application for European patent 173494 (published on March 5, 1986) Tapidcisnpi ei AI., application for European patent 173494 (published on March 5, 1986), /5 Meshbegdeg ei AI., PCT application UUO 8601533 (published on March 13, 1986); Kido et al. application for European patent 184187 (published June 11, 1986); Zapadap ei ai., U. Ittipoi. 137; 1066-1074 (1986); Kobripzop et al. application for International Patent UUO 8702671 (published on May 7, 1987); And in the name of AI., Rgos. Mai). Asad from the U.S.

BA 84: 3439-3443 (1987); Zip eyi aiYi., Rhos. May Asad All together. BOTH 84; 214-218 (1987); Ven. и Napomzh 5 I ape., AMTIVO0BIV: AGAVOKATOKU MAMOAI. (see above)). All instructions are incorporated into this description by reference. Anti-idiotypic (anti-1a) antibody is an antibody that recognizes an antigenic determinant, mainly associated with the antigen-binding center of the antibody. An idiotypic antibody can be obtained by immunization of an animal of the same species and genetic type (for example, a mouse line) as the source of TAB with a monoclonal antibody to which an anti-14 antibody is produced. Immunized animals can recognize and respond to idiotypic determinants of the immunizing antibody by producing antibodies to the specific idiotypic determinants (anti-14 antibodies). See, for example, US Patent 4,699,880, which i) is incorporated herein by reference.

The anti-idiotypic antibody can also be used as an "immunogen" to induce an immune response in a second animal producing the so-called anti-anti-idiotypic antibody. (This means that an anti-antiidiotypic antibody can be identical in its antigenic determinants to the original TAB, which induces an anti-idiotypic antibody. Thus, by using antibodies to the idiotypic determinants of TAB, other clones expressing antibodies of identical specificity can be identified.

Accordingly, monoclonal antibodies produced against 1C-binding proteins, their analogues or derivatives of the present invention or against рб551С, ро55ОО, РАБ-ОО, 1С, РА5Б-ОО, their analogues or derivatives, can be used for the generation of anti-idiotypic antibodies in the corresponding animals, such as mice of VACV/s. Spleen cells from these immunized mice are used to produce anti-14 hybrids secreting anti-14 tABs. In addition, these anti-14 tABs can be conjugated with a carrier such as hemocyanin of lymph snails (CIN) and used! for the "immunization of the second masses of VAI-V/s." The serum of these mice will contain anti-antiidiotypic 7-3) c antibodies that have the binding properties of the original tAbB, specific to the epitope of the above-mentioned 1C-binding protein, its analogs or derivatives, as well as roBb51C, ro55O0O, RA5B-1C or RAB-BO, their ;" analogues or derivatives.

Thus, anti-14 tAbBs have their own idiotypic probes, or "idiotypes!" structurally similar to the target probe, such as protein-o, CRV. -I The term "antibody" used in the present description also means intact molecules and their fragments), for example, such as Rar and (ab), which are capable of binding to an antigen. Rar- and

Fo Cabr')"-fragment! they represent a part of an intact antibody without an E-fragment, and are more quickly removed

Te) from the bloodstream and have less tissue-nonspecific binding than an intact antibody (Umani ei ai.,..

capes Honey. 24: 316-325 (1983). At the same time, it should be noted that ГРар-, К(аб)" - and other fragments used in the present invention can be used for the detection and quantitative assessment of 1C-binding proteins or р5о5б1с, ро5ОО, BAB-1С or РА5Б- ОЮ in accordance with the methods described in this application for intact antibody molecules. By the way, this is a fragment! obtained by proteolytic cleavage using enzymes such as papain (for the production of Rar fragments) or pepsin (for the production of

Kav)"-fragments). iF) It is considered that an antibody is "capable of binding" to a molecule if it is able to enter into a specific reaction with that molecule, as a result of which that molecule binds to the antibody.

The term "antibody" means a part of any molecule capable of binding to an antibody, which can be recognized by that antibody. The probes or "antigenic determinant" usually consist of chemically active groups on the surface of molecules, such as groups of amino acid residues or side polysaccharide chains, and have a specific spatial structure, as well as a specific charge. "Antigen" is a molecule or its part capable of binding to an antibody, and capable, in addition, of inducing in an animal the activation of antibodies capable of binding to the epitope of that antigen. Antigen b5 can have one or more subtypes. The term "antigen-specific reaction" means that this antigen can react with a high level of selectivity with the corresponding antibody, and cannot react with many other antibodies produced by other antigens.

Antibodies, including a fragment! Antibodies used in the present invention can be used for quantitative or qualitative detection of 1C-binding proteins or p551C, p55OrO, EBAZ-1C,

EAZ-0O in the sample, or to detect the presence of cells that express 1C-binding proteins of the present invention or the proteins robB1C, p5B5OO, RAB-1C, and RAB-0OO. Therefore, detection can be carried out with the help of immunofluorescence technique using fluorescently labeled antibodies (see below), in combination with methods of optical microscopy, flow cytometry, or fluorometry.

Antibodies (or their fragment), used in the present invention, can be used 7/0 in immunofluorescence or immunoelectron microscopy for the detection of 1C-binding proteins of the present invention or rb551C, ro50O, BAB-1C, EAB-0O. Ip ziz - detection can be carried out by taking a histological sample from the patient, and introducing the labeled antibody of the present invention into such a sample. The antibody (or its fragment) is preferably introduced by applying or coating a biological sample with the labeled antibody (or its fragment). Using this method, it is possible to determine not only the presence of 1C-binding proteins or ro51C, ro5OO, EBAZ-1C,

EA5B-OO, but also to estimate the distribution of these proteins in the examined tissue. It is absolutely obvious to any specialist that with the use of the present invention it is possible to modify any histological method of a wide range (for example, staining method) to implement such IP detection.

Indication of the analysis for the presence of 1C-binding proteins of the present invention or p551C, p55pOo,

RAZ-1C, RAZ-O0 are usually performed by incubating a biological sample, such as physiological fluid, tissue extract, freshly collected cells, for example, lymphocytes! or leukocytes, or cells that were incubated in tissue culture in the presence of a detectable labeled antibody capable of identifying 1C-binding proteins or rob51C, p55OO, RA5B-1C, EBAB-OO, and subsequent detection of that antibody by any of the available standard methods. with

A biological sample can be immobilized on a solid-phase carrier, such as microcellulose or another solid-phase carrier capable of immobilizing cells and particles! cells, or soluble i) proteins. This carrier can then be washed with appropriate buffers and then treated with a detectably labeled antibody in accordance with the present invention as described above.

The solid-phase support can be beaten, then washed again with buffer to remove unbound antibody. After that, the amount of bound label on the specified solid carrier or substrate can be determined by standard methods. and,

The terms "solid-phase support", "solid-phase support", "solid support", "solid support", Ge! "substrate" or "support" means any support or any support capable of binding to antigen or antibodies. Well-known supports or supports recognized are glass, polystyrene, polypropylene, ise)

Polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, cha gabbro and magnetite. By its nature, this carrier can be soluble to a certain degree or insoluble depending on the purposes of the present invention. The material for the carrier can have virtually any configuration, provided that the molecule immobilized on it is capable of binding to the antigen or antibody. So, for example, a substrate or carrier can have a spherical one in the form of balls) "

M cylindrical (in the form of the inner surface of a laboratory test tube) configuration, or represent ("With the outer surface of the rod. Alternatively, the surface of the carrier can be flat, such as a sheet, strip, etc. The preferred carriers are polystyrene balls. However , at the same time;" it should be noted that through routine experimentation, any specialist can independently choose suitable carriers for binding antibodies or antigens.

The binding activity of this antibody, described above, can be determined by certain methods. However, each specialist, by means of routine experimentation, can determine the working and optimal conditions of analysis for each specific case.

Me. In each specific case, the analysis can be performed in addition! other stages, such as co-washing, mixing, filtration, etc., if necessary.

One of the methods of obtaining a detectable labeled antibody of the present invention provides for the binding of this antibody to an enzyme followed by enzyme immunoassay (ELISA). This enzyme, in turn, can be subjected to interaction with the corresponding substrate, so that as a result of this reaction with the substrate, a chemical molecule or group was produced, which could be detected by the methods of spectrophotometry, fluorometry, or by visual observation. Enzymes that can be used! to obtain a detectable labeled antibody, include (but are not limited to) malate dehydrogenase, staphylococcal nuclease, steroid-5-isomerase, yeast (F, alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta -galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Antibody detection can be carried out by colorimetric methods that use a chromogenic substrate for the enzyme.

Detection can also be carried out by visual comparison of the degree of enzymatic reaction of the substrate with standards prepared in a similar manner.

Detection can also be performed using any second immunoassay. So, for example, radioactive labeling of antibodies or their fragment! can be detected by K-RTRase using 65 radioimmunological analysis (RIA). A good description of RIA is given (MUogk T. et al., I aroghagu

Tesppidnez apa Viospetivighu ip Moiesciag Vioodu; Moeii Noiiapa Rybiizpipda Sotrapu MM (1978) with a link to the chapter entitled "Ap Ipigodisiop (on Kadiottipe Avzzau apa Keiaiei Tesppidcevz", Spaga T.); the specified work is included in the present description by means of a link. A radioactive isotope can be detected using a gamma counter, scintillation counter, or autoradiography.

According to the present invention, the antibody can also be labeled with a fluorescent compound. However, if a fluorescently labeled antibody is exposed to light irradiation with the appropriate wavelength, the presence of that antibody can be detected thanks to fluorescence. The most common compounds used for fluorescent labeling are fluorescein isothiocyanate, rhodamine, phycoerythrin, picocyanin, allophycocyanin, o-phthalaldehyde, and 7/0 Ffluorescamine.

The antibody, for its detection, can also be labeled using fluorescent metals, such as 2E or other metals of the lanthanide series. Zti metal! they can beat you! with the antibody by means of groups that form chelate complex metals, for example, such groups as distylenetriaminepentaacetic acid (ETRA).

For its detection, the antibody can also be labeled by binding it to a chemiluminescent compound. The presence of such a chemiluminescent-labeled antibody can be detected by the presence of luminescence produced as a result of a chemical reaction. Examples of suitable chemiluminescent label compounds are luminol, isoluminol, heat-stable acridinium sulfate, imidazole, acridinium salts and acridinium sulfate, and oxalic acid.

Similarly, the bioluminescent compound can also be used to label the antibodies of the present invention. Bioluminescence is one of the types of chemiluminescence observed in biological systems, where the catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of bioluminescent protein can be detected by the presence of luminescence. Bioluminescent compounds suitable for labeling are luciferin, c luciferase, and zquorin.

The antibody molecule of the present invention can be adapted for use in i) immunometric analysis, known as a "two-layer" (or "two-stage") analysis or "szndvych" analysis. In a typical immunometric assay, a certain amount of unlabeled antibody (or its fragment) is bound to a solid support or to a solid carrier, and a certain amount of

The amount of detectable labeled soluble antibody is then analyzed for the presence and/or the amount of the ternary complex formed between the antibody immobilized on a solid-phase carrier, the antigen, and the labeling antibody is determined. Gee!

Generally and preferably, the immunometric analysis is a "direct" analysis, in which the antibody bound to the solid-phase carrier is first exposed to contact with the tested sample for the extraction of the antigen from the sample by means of the formation of a double complex "antibody on the solid-phase carrier - antigen" "' After incubation for an appropriate period of time, the solid support is washed to remove the remains of the liquid sample, including unreacted antigen, if any, and exposed to a solution containing an unknown amount of labeled "antibody" (which functions as a "reporter molecule"). After the second period of incubation, during which the labeled antibody forms a complex with the antigen bound to the solid phase by means of an unlabeled antibody, the solid carrier is washed a second time in order to remove the unreacted and labeled antibody. "» In the "szndvich" analysis of the second type, which can also be applied to the antigens of the present invention, the so-called "simultaneous" and "reverse" analysis is used. The simultaneous analysis provides for one stage of incubation, since the antibody bound to the solid carrier and labeled -And the antibody is added to the test sample at the same time. After the completion of the incubation stage, the solid support is shaken to remove the remains of the liquid sample and the unconjugated labeled antibody. Then the presence of the labeled antibody bound to the solid support is determined in the same way as in (Ce) "direct" analysis, description above.

In the "reverse" analysis, a solution of the labeled antibody is first added to the liquid sample, and then, after

An unlabeled antibody bound to a solid carrier is added to the mother of the corresponding incubation period. c» After the second incubation, the solid phase was washed in a standard way to remove the remains of the tested sample and the unreacted labeled antibody solution. Determination of labeled antibody bound to a solid carrier is carried out both in "simultaneous" and "direct" analyses.

New proteins of the present invention, after their isolation, identification, and characterization by any of the standard screening methods, for example, the method using yeast double hybrid, affinity chromatography, and other known methods, can be beaten, then produced! by standard recombinant DNA methods (see, for example, Zatgsok et al., 1989), in which suitable zukaryotic or prokaryotic host cells are transformed with appropriate zukaryotic or prokaryotic vectors containing protein-encoding sequences. Accordingly, the present invention also relates to expression vectors and transformed host cells. As mentioned above, these proteins also include their biologically active analogs and derivatives, and therefore, the present invention also refers to vectors encoding analogs of these proteins; and to transformed host cells producing these analogs. Production of the indicated proteins 65 is obtained by standard modification of proteins or their analogs produced by transformed host cells.

In its second aspect, the present invention relates to the use of a separate intracellular domain of p55-TME-K (p51C) or RGAB-K (GAZ-1C) or their so-called "death domains" (p55OYU or GAB-OO, respectively) as agents to enhance the effect of TME or HA5-K-ligand on cells, independently of each other (see Example 2). If in these cells, for example, in cancer or HIV-infected cells, it is necessary to generate a TME- or EAZ-K-ligand-induced cytotoxic effect, then the introduction of p51C, p55OO or RAB-OO into these cells can be done using the above-described method (see more p.(i)) using a recombinant animal virus (for example, cowpox virus). In addition, the present inventor can use native ro51cS, ro5OrO, RAB-1C or RHA5B-OO, their biologically active analogs, derivatives or fragments, and all of them can be obtained! methods, descriptions above.

Similarly, the present invention also applies to specific blocking

TME effect or RA5B-K-ligand action by inhibiting the activity of ro51C, ro5OO, BA5B-1C. or

EAB-0O, for example, by introducing an antisense oligonucleotide into the cells, leading to the blocking of the expression of ro51C, ro5OO, RAB-1C or EA5-0O.

The present invention also relates to pharmaceutical compositions containing recombinant vectors based on animal viruses, encoding proteins that bind to the intracellular domain of the TME or RAB receptor (including p5b51C, pb55OrO, BAB-1C and BA5B-OO), as well as encoding viral surface proteins capable of specifically bind to the surface proteins of target cells (for example, cancer cells), as a result of which, the vector guidance provides the directed insertion of 2o sequences of proteins that bind to the intracellular domain into the desired cells.

In its second aspect, the present invention relates, in particular, to the effects of self-association of the intracellular domain of the ro5-TME receptor (ro55-1C, see Example 2). An example of such effects, which are indirectly mediated by the binding of TME to its receptor, and which are imitated by the signal-transmitting activity of self-associating rbBb5-1C or their parts, is the induction of gene expression, with Coding 11-8.

Interleukin-8 (1-8) is a cytokine belonging to the subclass of chemokines, possessing i) pronounced chemotactic activity, and, as it has been shown, playing an important role in the chemotaxis of granulocytes and cells of other types associated with a number of pathological conditions | see. for example, Epao et al., 1994; Bekido et al., 1993; Nagada et al., 1993; and Regtisk et al., 19911. Tumor necrosis factor (TME) has useful activity, and therefore, it can be used directly to destroy tumor cells and virus-infected cells, or to enhance the antibacterial activity of granulocytes. However, as stated above, TME also has undesirable activity, and in those cases, it is desirable to block its activity, including cases when an increase in the dose of TME is used, for example, with anticancer, antiviral, and other antibacterial therapy. Cha

Accordingly, it is desirable to develop a method that would allow cells to regulate the activity of TME, or to obtain such a compound that would be able to mimic the beneficial activity of TME in cells or tissues that require special treatment.

In accordance with the present invention, it was discovered that the self-associating intracellular "domain of rob5-K (ro5-1C) can, in a ligand-independent manner, imitate a number of effects of TME, for example, with the "death domain" of rbo55-1C can induce cytotoxic effects! in cells, and rob5-1C can induce the expression of the 311-8 gene. Thus, using p5B-1C, it is possible to simulate the TME function by ;" site-directed mechanism, i.e., it is possible to introduce p55-1C only into those cells or tissues that need to be processed.

One of the examples of the above-mentioned methods can be the specific transfection (transformation) of tumor cells or malignant tissue with a DNA molecule encoding p55-1C or its fragment, which can not only induce cytotoxic effects! on these cells or tissues, but also strengthen them

Me. effect! by co-induction of 11I-8, which will lead to accumulation in the area of these cells or tissues with granulocytes or other lymphocytes, which, in turn, can contribute to the destruction of tumor cells or tissue. This method avoids the need to introduce large doses of TME associated with undesirable side effects. This can be obtained using standard techniques of recombinant DNA! different areas of 1C were studied and it was determined which area was responsible for each TME-induced effect. For example, it was established that the "death domain" is responsible for cytotoxicity (Example 2), and various other constructs containing parts of p5-1C have been obtained, which (together with part or all of the "death domain") may be responsible for other TMEs - effects, and the following can be used, (F, in a ligand-independent manner, after their self-association, for the purpose of inducing specified effects, such as inducing the expression of 11-8.

It should be noted that the sequence of rob5-1C, which is involved in the induction of other TME-associated effects (for example, the induction of 1I-8), may differ from the sequence responsible for cytotoxicity, i.e., it may not have it, or have only a part of it "domain of death", and have other "motives!" originating from other parts of the intracellular domain, or it itself can be a sequence of those other distinguishing features of the sequence (a sequence of the second "motive") participating in the induced second effects. 65 In accordance with what is described in detail above and below, it is possible to obtain an expressing vector containing the indication of the p55-1C region, its analogues or derivatives, and express it! in host cells, and then purification and analysis for their activity. In this method, a number of Rob-1C fragments possessing one or more TME-associated effects can be obtained, and this fragment! can be used in different ways for the treatment of pathological conditions of any number, for example, viral

Infections, bacterial infections, tumors, etc. In all specified cases, the specific activity of these fragments can be enhanced by co-introduction (or co-transfection) of the p55-1C fragment responsible for inducing the expression of the 1/-8 gene, which will lead to the desired chemotaxic activity of 11-8 and increased cell destruction or tissues that must be destroyed.

Thus, without resorting to the systemic introduction of TME, it is possible to induce its beneficial effects by the specific introduction of all rb5-1C or its fragment into cells or tissues intended for processing. rob5-1C can be specifically injected into cells or tissues for their destruction using any of the above-mentioned methods. So, for example, one of those methods provides for the construction of a recombinant animal virus (for example, originating from the cowpox virus), into the DNA of which the following two genes are introduced: a gene encoding a ligand that binds to cell surface proteins, specifically expressed cells, for example , the d9p120 protein of the AIDS virus, which specifically binds to some cells (CO4 lymphocytes and related leukemia cells); or a second ligand that specifically binds to cells carrying TME-K, and thanks to which the recombinant viral vector can bind to such TME-K-bearing cells; and the co-iodizing gene

V5Z-1C or ego fragment. Thus, the expression of a protein that binds to the cell surface on the surface of the virus will specifically direct the virus to the tumor cell or to another

a TME-K-carrying cell, as a result of which the sequence encoding p5o55-1C or its fragment will be, with the help of this virus, introduced into the cells, and the subsequent expression of this sequence in the specified cells will lead to an increase in the TME effect, which contributes to the death of tumor or of other TME-K-carrying substances Kkpetok, which must be destroyed, or inducing, for example, the expression of 1I-8, leading to the death of those cells. The construction of the specified recombinant animal virus can be carried out i) using standard techniques (see, for example, ZatrhookK et al., 1989). The second method involves the introduction of rb55-1C sequences or their fragments in the form of oligonucleotides, which can be absorbed! cells and zkspressirovan! they. The present invention also relates, in particular, to pharmaceutical compositions containing the above-mentioned recombinant viral vectors encoding p5b5-1Cb or its fragment, as well as those encoding a surface viral protein capable of specifically binding to surface proteins (such as target cells (for example , cancer cells), as a result of which the indicated vector provides insertion of the p55-1C sequence or its fragments into the indicated cell. ise)

In its second aspect, the present invention relates to new synthetic TME receptors, which are soluble and have the ability to oligomerize with the formation of a dimeric, and possibly higher order multimeric TME receptor molecule, each monomer part of which is capable of binding to a TME monomer . Native TME is a homotrimer containing three active TME monomers, each of which is capable of binding to one receptor molecule; whereas "natural TME receptors represent a single monomer, each of which is capable of binding only with one monomer of homotrimers of TME molecules. Thus, if TME binds to TME receptors on the cell surface, it is able to bind to three receptor molecules, which leads to the formation of TME receptor clusters, which obviously initiate the process of signal transmission, ultimately leading to the induction of the observed TME effects.

Although TME has many beneficial effects, such as the ability to destroy unwanted cells, for example, tumor cells or virus-infected cells, as well as to enhance the antibacterial activity of granulocytes, however, at the same time, TME has many undesirable

Me. zffectov, for example, those that lead to serious diseases, including autoimmune disorders, with rheumatoid arthritis, "transplant versus host" type rejection (graft rejection) and septic shock; and it is suspected that TME is the main cause of pathological destruction of tissues. TME can also cause excessive loss of body mass (cachexia) by suppressing the activity of adipocytes. In addition, even when TME is administered for the purpose of using its beneficial activity, for example, when treating various malignant tumors or viral diseases, the doses used

TME often turn out to be quite high and cause the patient a number of undesirable cytotoxic side effects, for example, destruction of healthy tissue.

Therefore, in all the above cases, where the effect of TME is unfavorable, it is desirable

F) use an effective TME inhibitor. Many TME-blocking agents have been developed, including soluble proteins that have the ability to bind to TME and inhibit the binding of TME to its receptors, thereby inhibiting the cytotoxic effect of TME. ER 308378, ER 398327 and ER bo 268925). However, these TME-binding proteins or soluble TME receptors are monomeric, which means that each of them is capable of binding to only one of the monomers of the TME homotrimer. Therefore, the blocking of the TME function by these proteins cannot be complete, since each TME molecule bound to a monomeric receptor has two more free TME monomers, which are capable of! contact with

TME receptors on the cell surface, and thus contribute to the adverse effect of TME on b5 K cells.

Therefore, in order to solve the problem associated with blocking the function of TME, in accordance with the present invention, a method was developed that allows the construction of hybrid proteins, namely, soluble oligomeric TME receptor), which have the ability to bind to at least two monomers native homotrimeric molecule! TME. According to this method, the indication is a soluble oligomeric receptor! TMEs bind to their TME-ligand with higher avidity than previously known soluble TME-binding proteins or receptors. So, for example, if soluble

The TME receptor of the present invention was obtained in the form of a dimer, so it is able to bind to two monomers of the TME trimer, which leads to a more complete and longer neutralization of TME due to a lower rate of dissociation of dimeric soluble receptors from TME. In addition, these soluble oligomeric receptors are also larger molecules than their monomeric counterparts, and therefore, from a pharmaceutical point of view, they have a great advantage due to the possibility that their elimination from the body will be slower.

The prerequisite for the development of soluble TME oligomeric receptors of the present invention was the discovery of the fact that the intracellular domain of the po5b5-TME receptor has the ability to /5 bamoassociation, and also the fact that in this intracellular domain (po5-1C) there is a section called " domain of death", which also has the ability of Kk self-association, and which directly, in a ligand-independent manner, can exert cytotoxic effects! on cells (see

Example 2). Using this self-association property of the intracellular domain (rb55-1C) and its "death domain", it is possible, with the help of standard recombinant DNA techniques, to construct a hybrid protein containing, basically, the entire extracellular domain of a TME receptor, such as the p/ 5-K or p5B-K, and preferably rob-K, and stitched with it, basically, the entire intracellular domain (ro5-1C) or the "death domain". Thus, it is possible to construct a new hybrid product, which has a TME-binding domain at one end, i.e., the extracellular domain of the receptor, and at the other end, an intracellular domain, or its "death domain", capable of self-association. Accordingly, with 2g5, the indicated product will have the ability to oligomerize due to self-association between its two (and possibly more) intracellular domains or "death domains", which will lead to the formation of i) oligomers (or, at least, dimers), containing at least two domains connecting with

TME.

In addition, in accordance with the present invention, it was also found that the GAB/APO1 c zo receptor has a self-associating intracellular domain, including a self-associating "death domain" that has certain homology with p55-1C and its "death domain" (Example 2). Therefore, in accordance with the present invention, it is possible to construct a soluble oligomeric TME receptor! by hybridization of the extracellular domain of the TME receptor (described above) with the intracellular domain or "death domain" of the GAZ/APO1 receptor. ise)

In both of the above-mentioned cases, the oligomeric TME receptors of the present invention are considered soluble only because they have a soluble extracellular domain of the TME receptor and a soluble intracellular domain or its "death domain" of either the p55-TME receptor or the receptor

EAB/AROT, that. they do not contain the transmembrane (insoluble) domain of any of the indicated receptors. "

A detailed description of the construction of the multi-specific TME oligomeric receptors of the present invention is given below, in the Example". However, it should be noted that after the construction

And the TME oligomeric receptors of the present invention may have a situation, not previously described, in which the extracellular domain of the TME receptor will be capable of Kk self-association, and such a situation may be undesirable, since it will prevent the ability of the oligomeric receptor to bind to two or more monomers of the homotrimeric TME molecules, or it may lead to less optimal binding of these TME monomers. Therefore, to avoid such situations, it is possible, with the help of standard recombinant DNA techniques, to modify the extracellular domain of the TME receptor, for example,

Me. by deleting or replacing one or more amino acid residues contained in the self-associating region in order to prevent possible self-association. Such a modification of the 5o extracellular domain of the TME receptor is, therefore, also part of the present invention, and the domains modified in this way are analogs or derivatives of the extracellular domain 4) of the TME receptor. Similarly, the self-associating intracellular domain (1C) or its "death domain" (00) of the rb55-K receptor or the BAB/APO1 receptor used in the TME oligomeric receptors of the present invention may also be an analog or derivative of the native domain, i.e., it can represent any modification of the rb55-1C sequence or its fragments, including the "death domain" (p55-OO), or any modification of the intracellular domain sequence (BA5-1C) or its

F) fragments of the GAB/APO1 receptor, including the "death domain" (GABA), provided that a self-associating product is formed as a result of silent modifications.

Similarly, after production and purification, the soluble oligomeric receptor! TME, their analogs or derivatives can be modified later! standard chemical means for the formation of salts and functional derivatives for the purpose of manufacturing pharmaceutical compositions containing the TME receptor! of the present invention as active ingredients.

To produce soluble oligomeric TME receptors of the present invention, DNA sequences encoding the extracellular domain of the TME receptor are obtained from existing clones of the complete TME receptor, and DNA sequences encoding the intracellular domain or its "death domain" of the TME receptor are obtained in the same way , as well as the intracellular domain or ego "domain of death"

RHAB/APO1 receptor (see Example 2 and Example 5). Then, the DNA sequence of the desired extracellular domain is ligated with the DNA sequence of the desired intracellular domain or its part containing the "death domain", and the thus obtained hybrid product is inserted (and ligated) into the corresponding expression vector under the control of the promoter or other expression-regulating sequences.

After creating such an expression vector, the vector is introduced into suitable host cells (i.e., the cells are transformed, transfected, etc. with the vector), and after the expression of the specified vector in the cells, the hydride product of the present invention is obtained, which is a soluble self-associating TME receptor molecule. Then the production of molecules in this way! 7/0 is isolated from host cells using standard techniques, and a soluble oligomeric receptor is obtained as the final product! THAT.

In a preferred variant, the hybrid sequences encoding the extracellular domain and the intracellular domain or its part are obtained using the RSK method using oligonucleotides with specific Kk data for the target sequences copied from clones 7/5 encoding the complete TIME receptor molecule. At the same time, it is possible to use the second method, which consists in isolating the necessary areas encoding the extracellular domain and the intracellular domain by means of restriction endonucleases followed by splicing using a known method to combine them, without modification or with modification of the ends of the restriction fragments to ensure the correct ligation of the necessary fragments of the receptor ( i.e., the extracellular and intracellular ego domains of each of their parts). The hybrid product obtained in this way is then inserted into the selected expression vector.

Similarly, the present invention also relates to soluble oligomeric EABZ/APO1 (GABZ) receptors, containing the extracellular domain of the RAB/APO1 receptor and the self-associating intracellular domain of rob-K (про5-1C) and the "death domain" (ро5ООЮ), or self-associating sh the intracellular domain of the EAZ/APO1 receptor (HA5-1C) or its "death domain" (GAABO), or any of their analogs or derivatives (see above). Below, in Example 5, a detailed description of the construction of those i) soluble oligomeric RGAbZ-receptors is given, where the existing cloned full-length sequence encoding the BAB receptor and the corresponding oligonucleotide were used as the starting material! for RSC production of the necessary extracellular and intracellular domains followed by their ligation to obtain a hybrid product, which was then inserted into the necessary expression vector. As described in detail above and below, for the production of the necessary soluble oligomers and

EAZZ-receptors can be used! both prokaryotic and zukaryotic host cells, after producing these receptors, they can be cleaned and used! as active ingredients in pharmaceutical compositions. ise)

Multiple indications of soluble oligomeric EAb-receptors of the present invention are intended! for effective blocking of the RHAbZ-ligand, which can also exist in the form of a trimer (similarly

TME, see below), moreover, each of the oligomeric receptors of the present invention is capable of binding to two or more RGAZ-ligands, thereby neutralizing their activity. It is known that GABZ-ligand is mainly a ligand associated with the cell surface, but it may also exist

M in soluble form. In any case, the oligomeric RAZ-receptor! of the present invention is able to bind to at least two monomers of that ligand, and thereby contribute to a more effective (than the monomeric EAb receptor) neutralization of the activity of the EAb ligand. EAZZ-ligand, and ;" therefore, the activation of the RA5 receptor plays a significant role in the occurrence of a number of pathological conditions, and in particular, conditions associated with liver damage (for example, apoptosis of hepatocytes),

Including liver damage associated with hepatitis, as well as autoimmune conditions, including destruction of -I (apoptosis) lymphocytes in HIV-infected patients | see, for example, Odazamaga et al., 1993; Spepa, et al., 1994). Therefore, soluble oligomeric EAbZ-receptors of the present invention are intended! for

Me. blocking the activity of RABZ-ligand, and can be used as an active ingredient in co-pharmaceutical compositions for the treatment of such RHA5-ligand-associated pathological conditions.

In a similar manner, the present invention also relates to soluble oligomeric receptors that have binding affinity for both TME and RAZ-K-ligand, and which represent the so-called 4) "mixed" oligomeric receptors TME-K/RA5B-K. This is a mixed oligomeric receptor! contain at least one TME-K extracellular domain and at least one RA5-K extracellular domain, which are combined in an oligomeric receptor due to the fact that each of them is attached to one of the above-mentioned self-associating rob51C, rozO0, GAZITS or GAZOO

This is a mixed oligomeric receptor! can be obtained by: a) production of any

F) multiple hybrid products that contain the extracellular domain of the TME-K receptor (p75-TME-K, or preferably p55-TME-K), linked to any one of the self-associating domains of p55-1C and GAB-1C or any one of the self-associating domains "domains of death" by RO5OO and BASE, or any ego or self-associating part, analogue or derivative; B) production of any of the above-mentioned hybrid products, which contain the extracellular domain of BAB-K, stitched with any one of the self-associating rbobB1C: BAB-1C, p55OO and RABA, or with any of its parts, analogues or derivatives; and c) mixing any of the TME-specific hybrid products a) with any of the

EAB-K-ligand-specific hybrid products B) and obtaining (after standard procedures of selection and 65 purification) oligomeric (dimers or oligomers of a higher order) receptors containing at least both extracellular domains of TME-K and EBAZ-K, which connections between themselves due to the ability to self-associate regions of 1C or BO connected with them.

To obtain mixed oligomeric receptors of multiple indications, it is possible to use the second method, which consists in the co-transformation of suitable host cells with the above-mentioned expressing vectors, one of which encodes TME-specific RA5B-K hybrid products, and the other encodes

EAB-K-ligand-specific GAZ-K-hybrid product!. After the expression of these different hybrid products in the host cells, the mixed oligomeric (TME-K/RAB-K) receptor! can beat receipts! through standard cleaning and selection procedures.

The value of these mixed affinity oligomeric receptors lies, first of all, in /0 TOM, that they neutralize both TME and RAB-K-ligand. in that case, if their excessive xndogenous expression is observed, or in that case, if after their zxogenic introduction, they are present in too large quantities. Recent observations point to the possibility that there is a synergism between the functions of PA5-K-ligand (generally associated with the cell surface) and TME-o (which may also be associated with the cell surface). Therefore, in some cases, it is desirable to neutralize both ligands in the same place on the cell surface, i.e., so that the receptor with mixed affinity can block the binding of TME to its receptor and binding

EAB-K-ligand and its receptor at the same time. Accordingly, receptors with mixed affinity can be used! as an active ingredient in compositions intended for the treatment of such compounds (see above) in which the activity of TME and GAZ-Klyganda is undesirable.

Similarly, in accordance with the above description relating to soluble oligomeric

TME-K, and BAB-K, and mixtures of TME-K/RAB-K - oligomers of the present invention, it is also possible to produce a soluble oligomeric receptor! of other types or mixed receptors originating from other types of receptors, and in particular, from any other members of the TME/MOR superfamily. In this case, the favorite extracellular domain of various receptors can be attached to the above-mentioned self-associating intracellular domains or their fragments, or to any other extracellular domains of members of the superfamily, which also have the ability to self-associate. at

Expression of any of the above-mentioned proteins of the present invention can be carried out in zukaryotic cells (for example, in yeast, insect or mammalian cells) using appropriate expression vectors. Any of the known methods can be used for these purposes.

So, for example, DNA molecules encoding proteins, obtained by the method described above, can be inserted into a specially designed expression vector! in accordance with the standard technique of

Ism. Zatrbgsok et al., 1989). Double-stranded DNA is ligated with plasmid vectors by homopolymeric chain extension or by restriction cross-linking using synthetic DNA linkers or by blunt-ended ligation techniques. For ligation of DNA molecules, DNA ligase is used, and undesirable parts of the connection are eliminated by treatment with alkaline phosphatase.

In order to express the desired protein, the expressing vector must also contain a specific nucleotide sequence capable of regulating transcription and translation, and which is attached to the DNA encoding the desired protein, so that gene expression with subsequent production of the desired protein could be carried out. In order for this gene to be transcribed, first of all, it is necessary that this gene was preceded by a promoter, which is recognized by RNA polymerase, and with which this polymerase binds, thus initiating the "transcription" process. There are various promoters, which act with different efficiency (stronger and weaker promoters).For prokaryotic and zukaryotic cells, different promoters are used.

Promoter), which can be used for the purposes of the present invention, are either constitutive (non-regulated) promoters, for example, the ip promoter of bacteriophage A, the B1a promoter of the rd-lactamase gene pVRZ22, and the CAT promoter of the chloramphenicol acetyltransferase gene pRRZ25, etc. or

F inducible promoters, such as prokaryotic promoters, including mainly the right and left (Ce) promoters of the bacteriophage dl (Pi, and Pv), the promoter of games, hesA, Ias/, Ias), otrE, and yes! E. soy, or syu 50 Shr-Ias hybrid promoter, etc. (SiiskK V.R. (1987)). To achieve high levels of gene expression in prokaryotic cells, in addition to the use of strong promoters that ensure the production of high amounts of mRNA, it is also necessary to use the site! binding to the ribosome to guarantee efficient translation of mRNA. As an example, the Schein-Dalgarno sequence (ZO sequence), preceding the initiating codon and complementary to the /3-end of the 165-RNA sequence, can serve as an example. o For zukaryotic hosts, there are many uses! different transcriptional and translational regulatory sequences depending on the nature of the host. These sequences may originate from viral sources, such as adenovirus, bovine papillomavirus, simian virus, or the like, where the regulatory signals are associated with a specific gene that has a high level of expression. As examples, the TK promoter of the herpes virus, the early promoter of the ZM 40 virus, the CA 4 yeast gene promoter, etc. can serve as examples. Regulatory sequences of transcription initiation can be selected so that they allow repression and activation of gene expression in order to modulate it.

A DNA molecule having a nucleotide sequence that encodes the hybrid protein of the present b5 invention is introduced into a vector that has a correspondingly attached transcriptional and translational regulatory sequence capable of integrating the desired gene sequence into the host cells. Cells that have been stably transformed by the introduced DNA can be selected by introducing one or more markers that allow the selection of host cells containing this expressing vector. The marker can provide phototrophy to auxotrophic hosts, resistance to biocide, for example, to antibiotics or to heavy metals, such as copper, etc. The selectable gene-marker can be either directly attached to the expressed gene DNA sequence, or introduced into the same cell by co-transfection. For optimal protein synthesis of the present invention, additional elements may also be introduced. Such elements are transcriptional promoters, enhancers, and termination signals. Vectors! zxpressiy 7/0 "DNA, introducing instruction elements, (descriptions of Okawata N. (1983).

In a preferred embodiment of the present invention, the introduced DNA molecule is inserted into a plasmid or viral vector capable of autonomous replication in the recipient cell. When selecting a specific plasmid! or a viral vector, important factors are: ease of recognition and selection of recipient cells containing this vector from cells that do not contain that vector; the number of copies /5 of the vector, which must be obtained in this specific host; and the fact whether it is desirable to use a "shuttle" vector capable of replicating in host cells of various types.

Preferred prokaryotic vectors are plasmids, for example, plasmids capable of replicating in E. soybeans, such as pVRZ22, SOYEI, pS101, pASUS 184, etc. see Mapiyaiz et al., 1982;

Zatrhook et al., 1989); plasmid! bacillus (Vasiiiis), such as pC194, pC221, pT127, etc. Ohuslap T. 19821; 2o plasmid! Zigeriyutusez, including kind 101 | Kepaai! K.U. and others, 19871; Zygeriotusev bacteriophages, such as 2C31

ISpayeg, KR. and others, in: 5ihiy Ipiegpaiopa! Zitrizvisht op Asiipotuseiyez Viokodu, (1986); and a plasmid!

Rezeidsotopaz (add., UR. et al. 1986; and Igaki K., 1978)). Preferred zukaryotic plasmids are VRU, cowpox virus, Zm 40, 2-micron ring, etc., or their derivatives. It's a plasmid! good lime! to specialists |Voivivipy O. and others, 1982; Vgoasp 9.K., in: Te Moiesshiag Vioiodu oi (She Ueavi sch ov Zasspagotusev; She Sesiye apa Ipregiapse (1981); Vgoasp U.K. (1982); VoPop O.R. ei ai., (1980); Mapiaiv

T., in: Seii Vioiodu; And Sotrgepepzvzime Tgeaiyize. MoiI.3; Sepe Ehrgeziop, (1980); and Zatgook eyi a!., 1989). and)

After obtaining the vector or DNA sequence containing the construct intended for expression, the DNA construct can be introduced into the corresponding host cell by any suitable method, for example, by transformation, transfection, conjugation, fusion of protoplasts, spectroporation, phosphate precipitation calcium, direct microinjection, etc.

Host cells used in the present invention can be prokaryotic or zukaryotic. Preferred prokaryotic hosts are bacteria such as E. soii., b

Streptomyces, Pseudomonas, Salmonella, Zeitaida, etc. The most preferred prokaryotic hosts are E. soya. Among the bacterial hosts, E. soya strain K12 294 ise) (ATSS 31446), E. soya X1776 (ATS 31537), E. soya MU3110 (E", lambda", prototrophic (ATOS 27325)) and other enterobacteria are of particular interest. , such as ZaItopeiIa iuUrpitigit or Zegitaiiz tagsezseps and various types of Rzepdotopase in such conditions, the protein will not be glycosylated. Prokaryotic host duties! beat compatible with the replicon and regulatory sequences in the expressing plasmid. "

The preferred zukaryotic hosts are mammalian cells, for example, human cells, monkeys, flies, and Chinese hamster ovary cells, since they provide post-translational modification of protein molecules, including its correct assembly or glycosylation in the necessary sites. Yeast cells are also capable of post-translational modification of peptides, including glycosylation. To produce the necessary proteins in yeast cells, there are a number of methods using recombinant DNA technologies, where a strong promoter and high-copy plasmids are used. Yeast cells recognize leader sequences on cloned gene products mammals, and secrete peptides carrying leader sequences (i.e., pre-peptides). After the introduction of the vector, the host cells are cultured in a selective medium used to select for the growth of vector-containing cells. The expression of the cloned gene sequences leads to (Ce) production of necessary proteins.

Purification of recombinant proteins is carried out by any of the known methods commonly used for

For this purpose, for example, by extraction, precipitation, chromatography, electrophoresis, or the like. c» The preferred method of purifying proteins of the present invention is affinity chromatography using monoclonal antibodies against the TME receptor immobilized on a gel matrix contained in a column. Therefore, unpurified preparations containing recombinant protein are omitted

Through the column. The protein binds to the column with the help of specific antibodies, while unpurified proteins will pass through the column. After washing, the protein is eluted from the gel by changing the pH or ionic strength. (name) As indicated above, the term "salts" refers to salts formed by carboxyl groups, and to acid salts of addition of amino groups of protein molecules!), which are obtained by known methods. 60 Salts of the carboxyl group include inorganic salts, for example, sodium and calcium salts, as well as salts formed with organic bases, for example, amines, such as trizethanolamine, arginine, or lysine. Acidic salts of addition are, for example, salts of mineral acids and salts of organic acids.

The term "functional derivative" used herein refers to derivatives 65 that can be obtained by modifying functional groups present as side chains on amino acid residues or M- or C-terminal groups by methods well known to those skilled in the art.

however, all such derivatives are included in the scope of the present invention, provided that they are pharmaceutically acceptable, i.e., they retain the activity of the original protein and do not impart toxic properties to the compositions containing them. Such derivatives are aliphatic compound zfir! or amide of carboxyl groups, and M-acyl derivative of free amino groups, or O-acyl derivative of free hydroxyl groups, formed by acyl groups (for example, alkanoyl or carbocyclic aroyl groups).

The term "fractions" used in this description refers to any part or fragment of a receptor (its intracellular or extracellular domains), or a protein that binds to the intracellular domain of a receptor, where the indication of a part or fragment! preserve the biological activity of this receptor or protein.

As indicated above, the present invention also relates to various pharmaceutical compositions containing a pharmaceutically acceptable carrier and various active ingredients described above! of the present invention or their salts, functional derivatives, or their mixtures. Zty 7/5 Compositions can be used in any of the conditions described in this application, for example, in conditions of overproduction of endogenous TME, for example, in septic shock, cachexia, "graft-versus-host" type rejection, autoimmune diseases, such as rheumatoid arthritis, etc. The method of administration of the compositions of the present invention can be any method acceptable for the administration of similar agents, and depends on the purpose and conditions of administration, so, for example, if the composition used is intended to inhibit the action of TIME, then it can be administered intravenously, for example, in the case of septic shock , or by local injection, for example, in the case of rheumatoid arthritis (for example, in the knee), or continuously, by infusion, etc. The compositions of the present invention can also be used in the case of TME intoxication caused by the introduction of too high amounts (overdose) of TME, for example, during anticancer or antiviral therapy. with

Pharmaceutical compositions of the present invention are obtained by mixing protein or its derivatives with physiologically acceptable carriers, stabilizers and fillers, and are made i) in the form of a dosage form, for example, by lyophilization in pharmaceutical bottles. The amount of the active compound required for administration depends on the method of administration, the specific disease, and the patient's condition. So, for example, a local injection for inflammation in the case of rheumatoid arthritis will require a smaller amount of the active ingredient per body weight than intravenous infusion in the case of septic shock. and,

Other aspects of the present invention will be obvious! from the following examples. b

In more detail, the present invention is set forth in the following examples, which, however, do not limit the scope of the present invention, and are illustrated in the accompanying drawings. ise)

Example 1

Cloning and isolation of proteins that bind to the intracellular domains of rob- and p75-TME receptors

The yeast two-hybrid system was used to isolate proteins that interact with the intracellular domains of the р5- and p75-TME receptors (р551С and р7б51С) (RBieiz 5 opo, 1989). The ZTA "0 two-hybrid system is a genetic analysis using yeast cells, which is carried out for the detection of specific "protein-protein" interactions by mime recovery

And zukaryotic transcriptional activator, such as SAI4, which has two separate domains: and? "DNA-binding domain" and "activation domain"; moreover, upon the expression of these domains and their binding to each other, a repair protein is formed (zZAI4, which has the ability to bind to

A multi-located activating sequence, which, in turn, activates the promoter, -I controls the expression of a "reporter" gene, such as Ias7 or NIZZ, and the expression of the reporter gene can be easily observed in cultured cells. In this analysis, geniuses for interacting candidates

Me. proteins were cloned in separate expression vectors. In one z-expressing vector, the sequence of one candidate protein was cloned in the corresponding phase with the sequence of 5p / DNA-binding domain of SAG4, resulting in a hybrid protein with the DNA-binding domain of SAII4, and in the second vector, the sequence of the second candidate protein was cloned in the same phase with this sequence of the CAI4 activation domain, resulting in a hybrid protein with the CA1/4 activation domain. Then there is a two-hybrid vector! were co-transformed into a yeast strain that has a "reporter" gene Ias7 or NIZZ, regulated by multiple CA14 binding sites. As a result, only transformed host cells (cotransformants!), in which two hybrid proteins capable of binding to each other are expressed, will express the reporter gene. In the case (F, using the Ias7 reporter gene, the host cells expressing this gene will be stained blue when X-da is added to the culture. Therefore, the blue color of the colonies will indicate that the two cloned candidate proteins are capable of interacting with each other using the indicated two-hybrid system, the intracellular domain of p5bB1C and p751C were cloned separately into the vector pOVTO9 (carrying the DNA-binding sequence CA 4; supplied by the company

SIGOMTESN, SHA, see below), resulting in hybrid proteins with a DNA-binding CA domain! 4 (in a similar way, the intracellular domain, RA5-1C and part of ro551C, namely 5500 were cloned! also in rOovtTO9 and used for the isolation of other 1C-binding proteins, see Example 3 below). For 65 Cloning of rob51C and r7b51C in rOVtTO, full-size encoding clones were used

cDNA sequences of roB5-TME-K, |Zspai et al., 1990) and p75-TME-K |Ztiiy et al., 1990), from which the intracellular domains were excised as follows: with the help of Esoki and zZai enzymes! po51c was excised, after which the EsokKiI-ZaiI fragment containing the po51C sequence was isolated by a standard method and inserted into the vector, restricted to it, in the region of the multiple cloning site (MO5), by enzymes

EsoKiI and ZaiYi. p/51C was excised using VerNi and Zaii enzymes, after which, the VZrNi-ZaiII fragment containing the p751C sequence was isolated by standard methods and inserted into the fragment

Klenova, as a result of which a fragment was obtained, which was inserted into the pOVTt9 vector, restricted by the enzyme zZtoi and zai).

Then the multi-hybrid (chimeric) vectors were co-transfected (separately, one 7/0 co-transfection was carried out using the p51C hybrid vector, and one using the p75 hybrid vector) together with the cDNA library from the human cells cloned into the vector rOAYUO ON, carrying the CA 4 activation domain, in the yeast strain NE?7c (all the above-mentioned vectors, pROVTO and rhodoO, carrying the cDNA library of Her cells, and the same yeast strain was obtained from Siopiesi and Aroghaigiev, Ips., OA, as part of MATSNNMAKEK Tmzho-Nubgid Zuziet, same RT1265-1). Co-transfected yeast was selected for its 75 ability to grow in a medium containing no histidine (Low-medium), however, growing colonies indicated a positive transformant. Then the selection of yeast clones was analyzed for their ability to express the Ias7 gene, i.e., for their G AC7-activity, by adding X-yes! in the culture medium, which undergoes catabolism with the formation of a blue-colored product with the participation of p-galactosidase, an enzyme encoded by the Ias/ gene. Thus, the appearance of pigeon colonies indicates the presence of an active Ias7 gene. In order to! gene Ias7 was active, it is necessary that! in the transformed clones, the CA14 transcription activator was present in an active form, namely, the DNA-binding domain

CAI4 encoded by one of the above hybrid vectors was correctly joined to the activation domain of CAG4 encoded by the second hybrid vector. Such a combination is possible only in the case when two proteins are connected to each of the SAI4 domains, capable of stable interaction of SU 25 (binding) with each other. Thus, Level 7 - and the white (AB) colonies, which were isolated, are colonies co-transfected with a vector encoding p5ob51C and a vector encoding a protein product originating from human Nei cells and capable of stably binding to ro51cC; or colonies transfected with a vector encoding P7B51C and a vector encoding a protein product of NegGa cells capable of stably binding to p751C. 30 Plasmid DNA was isolated from the above-mentioned yeasts Niz"', AS"-colony and introduced by co-electroporation into the HB101 strain of E. soybean according to the standard technique, after which the selection was carried out

And ampicillin-resistant transformants, i.e., transformants carrying the hybrid vector pOAOSsnH, which contains Atr'" and He? -coding sequences. After all, the instructions are transformative! "o represent clones carrying sequences encoding a newly identified protein, capable of binding to rob1c and p751C. Then, plasmid DNA was extracted from these transformed E. soybeans - and repeated testing was carried out by: a) re-transformation of these cells with the original hybrid plasmids containing the intracellular domain (hybrid plasmid roTVoe, carrying the sequence roB51C or p75-1C) by "insertion into the yeast strain NO7, as described above. As a control for co-transformation of C7Z70 with plasmids encoding p51C-binding protein or p/51C-binding protein, a vector carrying sequences carrying an irrelevant protein, for example, pAST-lamin or one pBTt9, was used. Then :z» co-transformed yeast was tested for growth on one Niz'-medium, or on medium with different levels of 3-aminotriazole; and

B) repeated transformation of plasmid DNA or initial hybrid plasmids, and control plasmids, described in (a), into yeast cells, host strain ZEUB26, and subsequent determination of GAS "7-activity (efficiency of formation of p-body, i.e. , development of blue color).

Me Result! The above-described tests showed that the nature of colony growth in Niz' medium is identical to the nature of (Se) IASO activity, which was evidenced by the coloration of the colonies, i.e., Niv" colonies were white as well as GAS". In addition, ACl activity in liquid culture (preferable culture conditions) was evaluated after

Transfections of hybrids containing the DNA-binding domain and the activation domain of SAG4 into yeast cells ZEU526, which have better I1AC7 inducibility by the transcription activator cA14 than the host cells of the yeast strain HE-7.

The result! implementation of several co-transfections presented below in Table 1, from which it is clear that protein detection is possible! bind to ro51C or p751C, namely, proteins, designation 55.11, which bind to ro51C; and 75.3 and 75.16, which are connected with r/51C. All indications of p51C - and i) p751cC-binding proteins are authentic human proteins encoded by cDNA sequences originating from the cDNA library of Neia cells, and assembled with the sequence of the activation domain of CAI4 in the plasmid pOAYUO OH in the above-described yeast or two-hybrid analysis system.

It is interesting to note that it was also found that the fragments of ro5b5b1c themselves, namely proteins, designated 55.1 and 55.3, are capable of! communicate with rob51C. This fact is also discussed below, in Example 2.

Table 1 b5

cDNA clones (see Example 3), bleached using two-hybrid systems! colony activation domain | liquid culture! Wevtls 00000000000000000000 IN VIA Establish 111111101 00000000

In the above Table, the following plasmids and hybrids encoding o

The DNA-binding domain of BAH 4 and the activation domain of BAH 4: so

Hybrids containing the DNA-binding domain: rovT9-1C55: full-size intracellular domain ro5-TME-K (ro51C) РаСтТ-lamin: irrelevant protein - lamin (Ce) roOVte: one vector

Zo rovtT9-1C75: full-size intracellular domain p75-TME-K (p751C) -

The hybrid containing the activation domain: 55.1 and 55.3 correspond to fragments of the P5B5-TME-K intracellular domain. 55.11: a novel protein associated with p55-TME-K « 75.3 and 75.16: novel proteins associated with p75-TME-K 7 70 Multiplex cloning of cDNA encoding novel p551C and p/51C-binding proteins, namely c 55.11, 75.3 and 75.16, were then sequenced using standard DNA sequencing methods. with Partial sequences of all protein-encoding sequences, readings: na

Fig. 1a-c, where Fig. 1(a) shows the cDNA sequence encoding protein 55.11; Figure 1(p) shows a partial cDNA sequence encoding protein 75.3; and Fig. (c) shows a partial 75 cDNA sequence encoding protein 75.16. Fig. 1(4) shows the amino acid sequence of protein 55.11, derived from the nucleotide sequence shown in Fig. 1(a).

Ge") However, it should be noted that the cDNA sequence coding for the 55.11 protein was also reported by |Kpap. and others (1992)). in their research devoted to cDNA sequencing of the brain and human, and aimed at the development of a new fast and accurate method of sequencing and physical and (95) 50 tenetic mapping of cDNA of the human brain. However, Kpap and others do not report anything about the functions or any other properties of the protein encoded by the 55.11 cDNA sequence, since this is a functional and other analysis! did not hit the goal of research Cap. and his employees.

Analysis and characterization of protein 55.11 a) General technique and material! 59th) cDNA cloning 55.11

GF) After analysis (for example, Northern analysis, see below) of the cDNA of protein 55.11, it was revealed that the above-mentioned cDNA of protein 55.11, cloned by the two-hybrid screening method, is only a partial

Protein cDNA 55.11, having a nucleotide! 925-2863 (see Fig. 1(a)), which encodes an amino acid! 309-900 (see

Fig. 1(4). The rest of the 55.11-cDNA (nucleotide! 1-924 (Fig. 1(a), which encodes the amino acid! 1-308 60 (Fig. 1(4))) was obtained by standard methods, namely, by PCR-cloning from cDNA- library of human fetal liver (for more details, see below). The complete nucleotide sequence of 55.11 (Fig. (a)) was determined in both directions by the dideoxy chain termination method. ii) Two-hybrid method of analysis for the expression of B-galactosidase!

The p-galactosidase expression test was carried out as described above, with the exception that instead of the 65 pOAYU-CH vector containing the SAI 4 activation domain, the pUR116 vector containing the UR16 activation domain was used. The numbering of the residues in the proteins encoded by cDNA inserts was the same as in the bank of these rAOAO-OH. Deletion mutations were produced with the help of RSK, and point mutations were produced with the help of oligonucleotide-directed mutagenesis | Kipkeyi, 19941. her) Northern analysis

Total RNA was isolated using TK1 KEASEMT (Moiesciag Kezeagsp Sepieg, Ips., Sipsippaiyi, OP.,

O.5.A.) were then denatured in a formaldehyde/formamide buffer, subjected to electrophoresis on an agarose/formaldehyde gel, and blocked on a Sepe ZSKEM Riiz membrane (Yuypopi, UMitipdiop, Oe.,

BA) in 10x 55 PE-buffer using standard technique. Blot! hybridized with partial cDNA 70 of protein 55.11 (see above, nucleotides 925-2863), subjected to radioactive labeling using a set for randomized priming (rseigzpdeg Mappeit Biospetisa, MappNeit, Segtapu), and washed under harsh conditions. Autoradiography was performed within 1 week. them) Expression of cDNA of protein 55.11 in Her cells! and binding of protein 55.11 to proteins representing a hybrid of glutathione-Z-transferase with rbo5-1C

We got a hybrid! glutathione-Z-transferases (351) with p5B5-1C (557-p551C) and with p5B51C, truncated below amino acids! 345 ((z5T-ro51C2345), and adsorbed on glutathione-agarose beads, as described below in

About 2 cm. also Zti(y 5: Sogsogap, 1994; Egapdiopi 5 Meei, 1993). 55.11 protein cDNA (nucleotides 1-2863, i.e., full-length 55.11 cDNA), P AC-55.11 and luciferase! expressed in Her cells and RIH AC-55.11 is a fragment of protein 55.11, extending between amino acid residues 309 and 900 (partial cDNA sequence of protein 55.11 (nucleotides 925-2863), initially cloned by the method of two-hybrid screening), and M-linked with RIH AC octapeptide (Razitap Kodak, Mem/ Namep, SI. O5A).

Expression of hybrid proteins was carried out using a tetracycline-regulated expression vector (NITA-1) in the clone of Neia cells, which expresses a tetracycline-regulated transactivator (see below, Example 2, and (Sozvzep 8: Vciaga, 19921). Metabolic labeling of expressed proteins with the help of su dv |58) Mei and |551 Suv (Oiropi, UMiItipdiop, Oe OB5A; Ategepat, Biskipud patvepige, Epdiapa), lysis of Nega cells, immunoprecipitation, and binding of labeled proteins with Z5T-hybrid proteins were carried out as described and) below ( Example 2), except that instead of 0.195 Mopidea P-40, 0.595 Mopidea P-40 was present in the cell lysis buffer. Immunoprecipitation of 55.11 and RGAS-55.11 was performed using rabbit antiserum (diluted 1:500) produced against the ZZT-hybrid protein containing the region "9 59.11, extending between amino acids 309 and 900, and a mouse monoclonal antibody against the octapeptide RIH AC (M2; Eazitap Kodak 5μg/ml cell lysate). and. o) Binding of protein 55.11 with ro55-1C in transformed yeast Ge»!

Because the study was undertaken to reveal nature! binding between 55.11 and rboBbbB1cS, and in particular, to reveal regions of those proteins involved in binding. For that, I used the two-hybrid technique described above, where there are different full-size and deletion mutants! p5o551c (see also below, Example 2) in the constructions of the "DNA-binding domain" was used as a "bait" for binding to the "prey", which is the incomplete protein 55.11, encoded in the constructions in which there is an incomplete 55.11 sequence ( residues 309-900, originally isolated) was added to the "activation domain" in the ZAG 4AO and MP1bAYU vectors. In addition, various deletion mutants were constructed! 55.11, which was ligated with the "activation domain" in the SAIG 4AO vector (for example, mutant 55.11, with only residues 309-680 and 457-900). The binding of various constructs with the "binding domain" and various constructs with the "activation domain" was studied in transfected yeast cells ZEU526b. ZME4 served as a control for the constructs with the "binding domain" and "activation domain", respectively; - and "empty" vectors (that is, not containing ba14 (rBAO-CH) and MRI16 (PMP1b6) served as a negative control for the constructs with the "activation domain"; and the "empty" (za14-vector (rOVTO) served as a negative control for the constructs with the "binding domain". The result! - and "nn" indicate the development of a bright color after 20-60 minutes after the start of the analysis, respectively (positive result! connection); and the symbol "-" means the absence of color after 24 hours after the start of the analysis (negative result!). An empty space in Table 2 means that no linkage analysis was performed). o now nn nya 60 b5 po tn NeYa -

PEZHEV because mine open a hundred VN Koantyn tya still that z y yy zv M ge ty /0 everyone will throw Vel "zhi tracking tov nty: EK Ya. y y ok pri M y pam ; her ev nkst, B sh vola

I'm like nn - I'm about She - oh

From the results presented in Table 2 above, it can be concluded that 55.11 is associated with rb55-1C in a site that does not belong to the "death domain" (residues 328-426) of r55-1C.

Protein 55.11 bound to the truncated p5o55-1C, from which the "death domain" was deleted (the construct from 206-328 in Table 2), more efficiently than to the complete p5-1C. In addition, this protein even binds to a more shortened C-terminus of p55-1C (construction 206-308), as well as to a construction from which the "death domain" and the membrane-proximal part of rob5-10 were removed (construction 243- 328). However, protein 55.11 did not (o) bind to the construct, which was truncated at the M-terminus to amino acids 266 (Table 2). These data indicate that the binding site for 55.11 is located in the region extending between residues 243 and 308 of p55-1C, and that the M-end of this binding site is located between residues 243 and 266. -

The transfer of the cDNA for the 55.11 protein from the original cloned construct of the "dog" containing the CA14 activation domain to the "dog" construct containing the P16 activation domain did not reduce the binding efficiency of the protein 55.11 to p55-1C (Table 2). Thus, the structure (or structures)) involved in this binding is obviously located in the 55.11 molecule and does not include the 55.11 fusion site with the activation domain. - c However, the binding of 55.11 with p55-1C does not occur even with limited truncation of the 55.11 protein in the ego C (55.11-construction 309-680)-end or M-end (55.11-construction 457-900). (Residue 309 is the first residue in protein 55.11, encoded by a partial cDNA clone, originally identified by two-hybrid screening).

The observed binding between 55.11 and p55-1C is apparently specific, since 55.11 did not bind to other proteins, including three receptors of the TME/MOE receptor family (p75-K, REAB/APO1, and bCO40), and to such proteins, such as lamin and cyclin O (these are not given). At the same time, it should be noted that from the other analyzed TME/M(OzE) protein receptors, those parts of them that contained the intracellular domain (Ce) were also tested: human EAB-K (residues 175-319), CO40 (residues 216-277 ) and p75-TME-K c 50 (residues 287-461), and none of them bound to 55.11 (data not shown). c) Northern analysis of RNA from several cell lines using 55.11 cDNA as a probe, and cloning of full-length 55.11 cDNA.

The researched cell lines are white cells Ne a, SEM, Chdigkai and Neros2, originating from epithelial carcinoma! human, acute lymphoblastic T-cell leukemia, acute T-cell leukemia, and hepatocellular carcinoma, respectively. First, the isolated 55.11 cDNA (nucleotide 925-2863) was used as a probe. Samples consisted of 1 µg of RNA per lane. The result of the Northern analysis of the readings in Fig. 2, on which the Northern blot is reproduced. Thus, it can be seen from Fig. 2 that as a result of Northern analysis performed for several cell lines using 55.11-cDNA as a probe, one hybridizing transcript, approximately 60 Zkr, was detected, which was larger than cDNA (2kb ) from the originally isolated 55.11-KDNK. Using an oligonucleotide primer that corresponded to the sequence of 55.11, the 5'-spanning sequence, the length of which was 11 cr, was cloned with the help of ROD. The sum of the lengths of the 5-spanning section and the initially cloned cDNA sequence is approximately equal to the length of the 55.11 transcript. Zkr-cDNA, which included both of these parts, was efficiently expressed in Nei a cells (see below) with the formation of a protein approximately 84 kDa long, which suggests that this Zkr-cDNA contains a translation initiation site.

a) IP migo - binding of protein 55.11 with 5T-hybrid proteins containing p55-1C regions

In order to make sure that 55.11 can really bind to rb55-1C, and that yeast proteins are not involved in this binding, the interaction of 5T-ro5-1C hybrid proteins produced by bacteria with the protein encoded by Zkr was investigated. -55.11 cDNA (full-length 55.11), and produced by transfected Neia cells. In this study, cDNA for full-length proteins 55.11, RIG AC-55.11 (residues 309-900 of protein 55.11, encoded by the initially cloned partial cDNA and fused, at its M-terminus, with the RI Ac octapeptide), and luciferase (control) were expressed in transfected cells of Neia, and metabolic methylation 95) May and 395) Suz. The following proteins were hybridized with O5tT: full-length p5-1C (55t-roB5-1C) and p55-1C, truncated at the C-end to amino acids! 345 (35T-r5o5-12345) in order to remove the "domain of death" (see Table 2). One 57 served as a control. Lyses of transfected cells were subjected to immunoprecipitation with antibodies against protein 55.11 in the case that full-length 55.11 was used for binding to Z5T-hybrid proteins; or with antibodies against the RGAS octapeptide in that case, if the RI As-55.11 hybrid product was used for binding to Z5T-hybrid 75 proteins. Proteins were analyzed by electrophoresis on a polyacrylamide gel with DSN (DSN-PAGE: 1095 acrylamide), and then subjected to autoradiography.

Presentation of autoradiograms at RysZA and ZV! of various DSN-PAGE gels, where Fig. ZA illustrates the binding of full-length protein 55.11 (55.11-70I) with various c5T-hybrid proteins; and Figure 3B illustrates the binding of Riad-55.11-hybrid product to various sZT-phybrid proteins. Figure ZA, on the far right lane, shows a control immunoprecipitate of cell lysates transfected with only one full-length 55.11 and immunoprecipitated with antibodies against 55.11 (antibodies o, 55.11). In Fig. 3B, the extreme lane on the right shows the control precipitate of lysates of cells transfected with only one BIH AsC-55.11 and immunoprecipitated with antibodies against GG AC (antibodies, RI A). Ge

Thus, it can be seen from Fig. ZA and ЗВ that the protein encoded by full-length cDNA 55.11 can (5) be expressed in cells No! and bind to hybrid proteins containing complete p5ob5-1C (o5T-p551C) or truncated p55-1C, which lacks most of the "death domain" (z5T-ro51C345) (Fig. ZA). The full-length protein 55.11 did not bind to one O5T (control). In a similar way, the protein expressed in Nei cells and encoded by the initially cloned partial cDNA for 55.11, Ge) hybridized with the octapeptide RIH AC (RI AC-55.11) bound to Z5T-r551C and o5T-r551C345, but did not bind to (51 (Fig. 3B). The above results are also considered as additional evidence (see above, p. (B)) that 55.11 is connected with the site of ro51C, the location of the "domain /F) of death", i.e., in the rb55-1C region, which is located closer to the transmembrane domain. with

In addition, the above-described study also demonstrated that in accordance with the present invention, it is possible to successfully produce antibodies against 55.11 (Fig. ZA). - e) Comparison of the deduced amino acid sequence of the human protein 55.11 with the sequences of related proteins present in lower organisms, and the peculiarities of the sequence of the protein 55.11

As mentioned above, in accordance with the present invention, the cDNA for the full-length protein 55.1 was cloned and sequenced (see the nucleotide sequence in Fig. 1(a), and from this cDNA sequence, the full-length amino acid sequence 55.11 was deduced (see the amino acid sequence in Fig. 1(4)). Studies of the sequences available in the "» data bank (SepVapktm/EMV1 BaiaVapk) showed that parts of the sequence of human 55.11 cDNA |reg.

MoMoTO3659, 19559 and RO128). and their muscle homolog |reg. MoMohX80422 and 7231147) were already determined! in the process of arbitrary sequencing of cDNA libraries. cDNA sequence (incoming Mo18247), which encodes a human protein of 596 amino acids, present in hepatoma cell cultures! HC10 is human, similar to the sequence encoding protein 55.11. However, the M-terminal part (amino acids 1-297) corresponding to that in 55.11 is missing in the hepatoma protein, and in addition, this protein differs from 55.11 in the regions corresponding to residues 297-377 and residues 648-668 in 55.11. Studies of these 50 sequences available in the data bank also revealed that the proteins present in Zasspagotusev sehemizide (yeast), Agaridorvziz (paiapa (plant Rezushka Thalia)) and Saepogparaiyiz eiedapz (helminth!) Thus, it is obvious that 55.11 evolutionarily conservative function. In yeast, there are two calcareous proteins (open reading frame UNRO27c and 5EMZ3), the DNA sequences of which are similar to the DNA sequence 55.11. The size of both of these sequences is also close to the size of 55.11. The sequence of UNRO27c became known only thanks to the sequencing of the genomic clone, then how the sequence of ZEMZ was cloned as cDNA. The site in 55.11, which is similar to the sites in ZEMZ, correlates in its identity with the sites in UNRO27c, although, obviously, greater similarity is observed between 55.11 and UNRO27c than between 55.11 and 5EMZ3. Available data (albeit only partial) about

DNA sequences for the proteins of Agaridorzis (paijapa and Saepogpabaiyis eedapz) clearly show that these 60 proteins are similar to 55.11, as well as the UNRO27s protein of yeast. Of these four proteins, the nature of which has been revealed to date, only one protein, namely, the yeast protein ZEMZ, which has limited homology with 55.11.5EM3 protein, was identified as the yeast equivalent of p112 subunits of the proteosome activator! 205

I (the proteolytic central part (core) of proteosomes 265 Kespvzievipeg et al., 1993; YuOemagipo et al. 1994) |M.R.

SiPetpizop and M. NoskKzigazzeg, personal communication. 65 Figure 4 schematically illustrates a comparison of the derived amino acid sequence 55.11 of a human with the sequences of the above-mentioned related proteins present in lower organisms. The sequences compared in Fig. 4 are amino acid sequences predicted for: 55.11-kDNA (see Fig. 1(a)); open reading frame (UNRO27s) in the cosmid originating from the 8th chromosome! Zasspagotusez segemiviae (nucleotide! 21253-24234, accession number 10399); protein cDNA

Zasspagotusez sehemizide (admission number 06321); partial cDNA protein of the plant Agabridorvzis (Paiyapa (rezushka

Talya) (admission number T21500), and partial cDNA of nematode protein! Saepogpabayiiiz eiedapz (admission number 27396).

In Fig. 4, the sequence "KEKE" in 55.11 is marked by a solid line, and the sequence AUAHOB(X) in |. marked with a dotted line. The comparative analysis of the primary structures of the sequences was carried out using the RI EOR and RKETTUVOH programs of the SO package. "Bresha", an introduction to maximize 7/0 comparisons, designations! points

As for the distinguishing features of different sequences, or motifs present in the sequence of 55.11 human, the following observations were made. In the protein encoded by the 55.11 sequence, with the exception of the repeating sequence "KEKE", which is between Guz 614 and sy 632 (highlighted in Fig. 4), no "motifs" of a conservative amino acid sequence were found. Such "KEKE" sequences, which are present in many proteins, including proteasomal subunits and proteins - "mentors" (sparegopips), which can stimulate the association of protein complexes | The sequence AUASB(X) and HER 11 appears in the sequence of protein 55.11 twice (in sites 479 and 590, see Fig. 4), however, any functional role of this sequence has not been established.

C) Distinctive features of the sequence of the p55-1C region involved in binding to protein 55.11

As described above (see paragraphs (B) and (4)), protein 55.11 binds to the rb55-1C region located between residues 243 and 308 (the M-end of this binding site is located between residues 243 and 266), and located above "death domain" and closer to the "transmembrane" domain of p5-MTE-K. This region in p55-1C, with which protein 55.11 binds, has a high content of proline, serine, and threonine residues. However, this region does not contain enriched proline "motifs" "KRMI and KRM2, present in some other cytokine receptors |O' Mea! Uciee, 1993). In the region that extends between residues 243 and i) 266, and the deletion of which leads to the absence of binding of robB-K, p 55.11 (see above, paragraphs (a) and (B), and Table 2), two serine residues and two threonine residues are followed by proline residues, which makes them potential sites for phosphorylation by MAP kinase, СОС2, and other proline-independent kinases IZedeg and Kterv , 1995), phosphorylation in zt the site of receptors can affect their binding to protein 55.11. and,

Taking into account the above data related to protein 55.11 and its connection with p5o5-1cS, Ge! it is possible to conclude that in accordance with the present invention a new protein has been discovered that binds to a specific region located above the "death domain" p55-1C. Therefore, the binding must have an effect on TME mediation. activity, with the exception of inducing cell death. As previously shown, the region to which 55.11 binds is involved in induced synthesis! nitrogen oxides

ITapadiia et al., 1993), and, apparently, participates in the activation of neutral sphingomyelinase by tumor necrosis factor (Miedtapp et al., 1994). Thus, it is possible that the association (binding) of protein 55.11 with the intracellular domain of ro5-TA/R-K, (rb551C) exerts an influence or participates in (i) signal transmission for the above-mentioned or other TME effects; (ii) folding or processing of the protein (as assumed based on the similarity of 55.11 with the proteasome subunit! 265); or (ii) regulation of activity or expression of P55-TME-K.

Example 2;" The ability to self-associate the intracellular domain of the p5-TME receptor (p551C), its ability to induce cell death, and its other distinguishing features and functions; and the intracellular domain of RAB/APO1 related receptors. - And as indicated above, in Example 1, it was found that the intracellular domain of roB5-TME (roB51c) is capable of binding to itself, and in addition, it was established that the fragment! ro51C, namely, proteins me) 55.1 and 55.3, also capable! to communicate with ro51s.

Ge) It is known that the binding of TME with p55-TME-K leads to a cytocidal effect on cells carrying this receptor. In addition, antibodies against the extracellular domain of that receptor can themselves stimulate that action in accordance with the effectiveness of cross-linking of the receptor with those antibodies. 4) In addition, mutational studies |Thagiadia et al., 1993; Vgaskerysp et al., 1992) showed that the function of p55-K depends on the integrity of its intracellular domain. Therefore, there are reasons to assume that the initiation of signal transmission for the cytocidal effect of TME occurs as a result of the association of two or two Several extracellular domains of roo-K (rovb-1C), which induces receptor aggregation. The results obtained in accordance with the present invention confirm this conclusion and indicate that the expression of the intracellular domain of p55-K in cells without a transmembrane or intracellular domain stimulates the death of those cells. It was shown that such a free intracellular domain p55-K is capable of self-association, which apparently accounts for their ability to function independently of the TME.The fact that signaling by full-length p55-K depends on TME stimulation is thought to reflect the activity of the transmembrane or extracellular domain of the receptor, which promotes reduction or prevention of this self-association.

The ability of the intracellular domain of rob5-K (p55-1C) to self-associate was discovered by chance when trying to clone an effector protein that interacts with this receptor (see above, Example 1). 65 For these purposes, the more described "two-hybrid" method was used. However, it was found that, in addition to the new protein, 55.11 associates (binds) with p5obB-1C, and it was also found that the three second cloned cDNA cells of Nea contain cDNA sequences encoding parts of the intracellular domain of p5ob5-K, which means that p55-1C has the ability to self-associate. Two of those clones were identical and contained an insert that encodes an amino acid! 328-426 (this is a clone! white designations

CloNoM 55.1, encoding protein fragment 55.1 p551C). The third clone contained a longer insert, coding for amino acids 277-426 (this clone was designated as clone 55.3, which corresponds to the protein fragment 55.3 rb551cS).

In addition, we evaluated the interaction between two p5oBb1cS chimeras produced in bacteria, one of which was hybridized with maltose-binding protein (MBB), and the other 70 was hybridized with glutathione-5-transferase (551). It's a whim! they were constructed, cloned and expressed by standard methods. After their expression, the self-association of the intracellular domain p5bB-K (ro51C) was evaluated by analyzing the interaction of the various bacterially produced chimeric proteins 5T1-1C55 (Mi-51kDa) and MVR-1C55 (Mi-67kDa) with each other. Therefore, equal to the number of chimeras! Z1T-1C55 (samples of lanes 1-4 in Fig. 5) or one O5T (samples of lanes 5-8 in /5 Fig. 5) were immobilized on glutathione-agarose beads (Zidta), and then incubated with the same amount of MVR- 1C55-hybrid protein in one of the following buffer solutions: i) buffer 1 (20mM Tris-HCl, pH7.5, 100mM KIS, 2mM Sasi», 2MM Maosi», 5mM OTT, 0.295 Triton X100, 0.5MM

РМ5РЕ, 595 Glycerin). This buffer was used for samples of lanes 1 and 5 in Fig. 5. her) buffer 1 containing 5 mM EOTA instead of Masi". This buffer was used for the samples of lanes 2 and 2

Fig". her) buffer 1 containing 250 mM instead of 100 mM KS. This buffer was used for! samples of tracks C and 7 in Fig. 5. them) buffer 1 containing 400 mM instead of 100 mM KSI. This buffer was used for the samples of lanes 4 and 8 in Fig. 5. high school

After incubation with rotation for 2 hours at 42C, beads were washed with the same buffers, and then boiled in DSN-PAGE-buffer with subsequent electrophoresis in PAGE. The proteins on the gel were subjected to

Western blotting on a nitrocellulose membrane, which was then stained with a polyclonal antiserum against MBR. The indicated staining Western blot is shown in Fig. 5 (samples in lanes 1-8, described above). co

It can be seen from Fig. 5 that chimera ro510-O5t binds to chimera ro51C-O5t (lanes 1-4) regardless of the presence of divalent cations, and even at a sufficiently high salt concentration (0.4 M KSI). Hence and. it is possible to conclude that ro51C has the ability to avid self-association. Gee!

To evaluate the functional role of this ability of rb55-1C to self-associate, we attempted to express rb5-1C in the cytoplasm of cells that are susceptible to the cytocidal action of TME. at

Taking into account the fact that rb55-1C, in turn, can be cytotoxic, hm! vibrated it - an inducible type of expression, used for this purpose a recently developed tightly regulated tetracycline-controlled expression system of a mammal (Sozzep and Vociaga, 19921. Expression of ro5-1C led to mass cell death (Fig.b, right panel). Dying cells are observed loosening of the cell surface, as occurs during TME-killing of cells. Transfection of the pHO5-1C construct into cells in the presence of tetracycline, which markedly reduces the expression level of PO) with pHO10-3-regulated constructs up to a 102-fold decrease, still leads to the death of some cells, although to a much lesser extent than that observed in the absence of tetracycline (Fig. b, left panel). In contrast, cells transfected with a control construct containing luciferase cDNA did not show any signs of death (result! not given).

The ability of p5B5-1C to stimulate cell death, when it is expressed without the transmembrane or -I extracellular domains of the receptor, once again indicates that this domain is involved in signal transmission. In addition, this fact indicates that none of the other parts of the receptor play a direct role in such signal transmission. My studies of the effect of mutations, including mutations (Ce) considered in the present invention, on the function of p55-1C have shown that the most important region for this function is the region extending between amino acid residues 326 and 407. This region shows a noticeable similarity with the sequences of intracellular domains of two others

Fe receptors, which are evolutionarily related to the p5-TME receptor, namely, the EAZ3 receptor (Yi et al., 1991; Oeepyt et al., 1992), which is also capable of transmitting a cytocidal signal; and СО40 - receptor (Ziatepkomis et al., 1989), which promotes cell growth; therefore, the sequence of this region obviously contains a conservative motif that plays one of the main roles in signal transmission. Since there are no similar known "motifs" characteristic of enzymatic activity, it is obvious that this region transmits the signal in a mediated way, i.e., it probably serves as a "docking-acceptor" site for signal-transmitting enzymes or for proteins that transmit stimulating signals for these enzymes. All p55-1C, EAZ receptor and CO 40 can be stimulated by antibodies against their extracellular domain. This stimulation should, as it was shown, correlate with the ability of antibodies to cross-link with receptors. Therefore, it is obvious that signal transmission is initiated as a result of the interaction of two or more intracellular domains induced by the aggregation of extracellular domains. Participation in such an interaction of receptors with subsequent signal transmission was also established by research (Vgaskerys et al., 1992), which showed that 65 zxexpression of receptors deprived of their functions by mutation of their intracellular domain had a "dominant negative" effect on the function of co-expressed receptors normal receptors. Aggregation of rob-K in response to the action of TME suggests that it occurs passively and is due only to the fact that each of the molecules of TME, which is a homotrimer, can bind to two or three receptor molecules. However, the data obtained as a result of the implementation of the present invention give grounds to assume that this process proceeds somewhat according to the second type.

The tendency of p55-1C to self-associate indicates that this domain plays an active role in its induced aggregation. In addition, this activity of p55-1C is apparently sufficient for the initiation of signal transmission, since when it is expressed independently of the rest of the receptor molecules, it can stimulate cell death in the absence of TME or any other external stimuli. However, when 7/0 is expressed as a full-length receptor, p5-TME-K does not transmit a signal if it is not stimulated by TME. Therefore, it can be concluded that upon activation of the ro5-TME receptor, TME actually overcomes some inhibitory mechanisms that prevent the spontaneous association of intracellular domains, and that the inhibition is due to the binding of rob-1C to the rest of the receptor molecule. Inhibition may be due to the orientation given to the intracellular 7/5 domain by the transmembrane and extracellular domains; binding of some other proteins to the receptor; or, most likely, by limiting the number of receptors that can be located in the plasma membrane. It goes without saying that this regulation mechanism must be sufficiently effective, since, according to some estimates, the binding of even just one TME molecule to a cell is sufficient to stimulate its death.

Spontaneous signal transmission, regardless of the ligand, can lead to significant disruption of the process regulated by this receptor. The most famous example of this is the cessation of regulation of growth factor receptors. So, for example, in the uncontrolled growth of tumor cells, an important role is played by mutations, as a result of which the initiation of signal transmission becomes spontaneous, for example, mutations that cause spontaneous aggregation of receptors. It is well known that TME effects, with their excessive MnD induced play an important role in the pathology of many diseases. The ability of the free intracellular domains (rbo51C) of the p55-TME receptor to transmit a signal independent of TME may contribute to such (i) pathological phenomena. It is quite possible, for example, that the cytopathic action of some viruses and other pathogens is not due to their direct cytocidal activity, but to proteolytic cleavage of the intracellular domain of the p5-TME receptor, leading to a TME-like cytotoxic effect. с зо For a more precise identification of the region (or regions) in ro551C responsible for its ability to self-associate, and therefore for its ligand-independent cytotoxicity, as well as to determine whether there are other related members of the TME/MOR receptor family ( for example, EAB-K) intracellular domain! with Ge! the ability of Kk self-association and Kk ligand-independent action, careful research was carried out, the description is below. ise) a) General method and material! ii) Two-hybrid screening and two-hybrid test for p-galactosidase expression

cDNA inserts encoding p5o5-1C and its deletion mutants, EAB-1C and various other proteins (see

Table 3) was cloned using RSC, from full-length cDNAs cloned earlier in our laboratory, or from commercially purchased cDNA libraries. Expression of p-galactosidase in yeast "Ireporter strain EU526; Vapei et al., 1993), cDNAs transformed with the instructions of rOVT-9 and SHO vectors with rOAO-CH (constructs with a DNA-binding domain (OBO) and an activation domain (AB), respectively) were evaluated using a test in a solution | , 1983); and also performed an analysis on filters for "" qualitative evaluation of the same results (not shown). Two-hybrid screening (Riveids and Zopa 1989) of the purchased SAI 4-At1-labeled cDNA library of Nei a cells (Siopiesp, Rayu A, Sa., О0.5.A.) for proteins that bind to the intracellular domain of rob-K (p55-1C) were performed using -I reporter yeast strain HE7c in accordance with the manufacturer's recommendations. The positivity of the isolated clones was assessed by (a) prototrophy of the transformed yeast in relation to histidine

F is cultivated in the presence of 5 mM Z-aminotriazole; (B) expression of β-galactosidase; and c) test for (Ce) specificity (interaction with Z5ME4 and lamin merged with ZAG 4 JUVO). o 50th) Self-association of migo ro5-1C-hybrid proteins produced in bacteria

Glutathione-Z-transferase (551) and the hybrid protein "glutathione-Z-transferase - p5B5-1C (55T-p55-1C) were produced as described in the literature (Egapdiop and Meei, 1993; Alizivei et al., 1994 ). Maltose-binding (MBR) hybrid proteins were obtained using the rMAISSKkt1 vector (Mem/Epdiapa Violyar5) and purified on a column with amylose resin. The interaction of MBRR and c5T hybrid proteins was studied by successive incubation of glutathione-agarose beads with 35T and MVRR hybrid proteins proteins (5 μg of protein/200 μl of beads; the first incubation was carried out for 15 minutes, and the second for 2 hours, both at 42C). Incubation with MBR-hybrid proteins was carried out in a buffer solution containing 20 mM imi) Tris-HCI, pH 7, 5, 100 mM KS, 2 mM Sas, 2 mM MoSiI, 5 mM dithiothreitol, 0.295 Triton X100, 0.5 mM phenyl-methyl-sulfonyl fluoride and 595 (by volume) glycerol; if necessary, in the same buffer containing 0 ,4M KSI, or 5mm EOTA instead of MosSi". Associating ICBM-hybr Some proteins were evaluated using polyacrylamide gel electrophoresis with DSN (1095 acrylamide) of proteins bound to glutathione-agarose beads and subsequent Western blotting. Blots were probed using rabbit antiserum against MBR (produced in our laboratory) and goat antirabbit immunoglobulin conjugated with horseradish peroxidase. 65 ii) Induced expression of the p5-receptor and its fragments in Her cells

Nea cells expressing tetracycline-controlled transactivator, development of bozep and

Vtsiaga (clone NITA-1 | Sozzep and Vtsiaga, 19921), cultivated in modified Dulbecco medium

A needle containing 1095 fetal calf serum, 100 µg/ml penicillin, 100 µg/ml streptomycin and

O.5 mg/ml neomycin. cDNA inserts encoding ro5b-K or its fragment were inserted into the tetracycline-control-expressing vector (RONO 10-3, kindly provided by E. Vyaga).

Cells were transfected with an expression construct (5 μg of DNA/bsm dish) using the calcium phosphate precipitation method (Alizivei et al., 1994). The result! Short-term expression of transfected proteins was evaluated at appropriate time intervals after transfection in the presence or absence of tetracycline (Imkg/ml). Clones of cells stably transfected with human ro5b5-1C cDNA in the rinA 10-3, 7/0 vector were confirmed by transfection of cDNA into NETA-1 cells in the presence of tetracycline together with a plasmid carrying resistance to hygromycin, and subsequent selection on clones. resistant to hygromycin (200 μg/ml). CkDNA expression was obtained by removing tetracycline, which in other cases was constantly present in the medium for cell growth. them) Evaluation of TME-like effects stimulated by induced expression of R5Bb5-K and ego 7/5 fragments

The effect of induced receptor and TME expression on cell viability was assessed by the neutral red absorption method (MaMPasy, 1984). The induction of the expression of the 1I-8 gene was assessed by the method

Northern analysis. RNA was isolated using TRI REASEMT (MoiYescshiag Kezeagsp Sepieg, Ips.), then denatured in formaldehyde/formamide buffer, subjected to electrophoresis on a 2o agarose/formaldehyde gel, and blocked on a Sepe bsgeep Reese (Yuy Rop) membrane in 100XxRE-buffer using a standard techniques Filter! hybridized with the 11-8 cDNA probe (Maizizpita, et al., 1988) (nucleotides 1-392), subjected to radioactive labeling using a set for random priming (Voephipdeg Mappeit Viospetisa, Mappeit, Segtapu), and incubated under strict conditions in in accordance with the manufacturer's instructions. Autoradiography was performed within 1-2 days. c m) Evaluation of TME receptor expression

The expression of the TME receptor in samples of 1x109 cells was evaluated by measuring the binding of TME, labeled with |22II according to the previously described method using chloramine-T | Neutapp and Umaias, 19871. EHIZA analysis was also carried out, as described in the literature for the quantitative assessment of soluble TME -receptors | Adegka et al. 1991) with the exception of the fact that for cell lysis (7 Ωcl/10 Incl.) and i) for dilution of the tested samples, VIRA buffer was used (10 mM Tris-HCl, pH7.5, 150 mM Mass, 195 so

MP-40, 196 deoxycholate, 0.190 DSN, and imM EOTA). The soluble form of p55-K isolated from urine served as a standard. Me) o) Mutational analysis of the intracellular domain of rbob5-K (rb55-1C) to determine the regions of p5B5-1C involved in self-association

As mentioned above, p5-1C has the ability to self-associate, and there are such areas p55-1C, c. which you yourself are capable of! connect with the full-size p55-1C. In particular, one of the fragments of ro55-1C (designated protein fragment 55.1 in Example 1, see above) was identified, which is capable of strong binding to full-length ro55-1C; however, after sequencing that fragment, it was established that it is located between amino acid residues 328 and 426 of the p55-TME receptor and is located in p55-1C. In addition, it was established (see below) that the above-mentioned region, namely, fragment 55.1 itself o) c is capable of self-association and stimulation of cytotoxic effects on cells. Therefore, this region p55-1C "" was called the "death domain", which is located between amino acid residues 328-426 of human p5B5-K ", and in all likelihood, consists of amino acid residues located between approximately residues 328 and 414.

The fact that the "death domain" in rb55-1C is capable of self-association was discovered by chance. After - screening of the cDNA library of Nea cells using the two-hybrid technique (see above, Example 1) in

In order to identify sequences encoding proteins that bind to the intracellular domain of this receptor, several clones (for example, 55.1 and 55.3) were found among the cDNAs, the products of which specifically bind to the hybrid protein "intracellular domain - ZAGA OVO" 20 coding regions of the p55-E intracellular domain (p55-1C; marked with asterisks in Table 3).

With the use of a two-hybrid test to assess the degree of specificity in the self-association of ro5-1C and c" for a more accurate definition of the area involved in this self-association, whites were obtained! the following data (Table 3): a) self-association of ro55-1С is limited to the area located in the "domain of death". Eh

The M-terminus is located between residues 328 and 344, and its C-terminus is located near residue 404 and slightly higher than the C-terminus (as indicated in the literature) of that domain (residue 414). (B) Deletion of the proximal membrane

GF! the p55-1C region, located above the "death domain", promotes self-association, which gives reason to assume that this region exerts an inhibitory effect on association. c) MyIshiny o rob-1C is capable of self-association, as well as of connection with the "death domain" of human rb55-K. 4) Studies of the self-association of the intracellular domains of three other receptors belonging to the family of 60 TA/g/SvB receptors, namely, the EAB/APO1 (HA5-K) receptor, CO40 (Gryeidz and Zopd 1989), and the p75-TME receptor

Iztiy et al., 1990) showed that HA5B-1C, which transmits a cytocidal signal with the help of a specific sequence - a "motif", related to the "death domain" of rbo5-K, is capable of self-association, and to some extent about the association of rboB -1C. However, CO40-1C, which provides the transmission of growth-stimulating signals (even despite the fact that it also contains a sequence similar to the "death domain"), and p75-1C, which is structurally not similar to p55-1C, do not have a tendency to self-associate and do not associate with ro5-1C or EA5B-16.

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In (Se) 35 In the above-mentioned Table C, a quantitative analysis of the interaction of

SAI 4-hybrid constructs, which include the following proteins: the intracellular domain of human p55-K and its various deletion mutants! (remnants of white numbering!, as in the work of I oeizspeg et al., (1990)) and designations here as ZE 10 Mo:25; muscle intracellular domain of robB-K (residues 334-454; numbered as (in the work of Soodm/ip et al., (1991)); muscle RAB/APO1, GA5-1C, residues numbered as!

In the work of UUaiapare-Rykapada et al., 19921; person СО40 (СО040-10, 216-277, remnants of numberings, as (in work with Ziatepsomis et al., 1989)); and people p/5-TME receptor (p75-1C, 287-461, the rest of the numberings! as | in work

Ztik et al., 19901). ZM E1 and ZME4 were used as positive controls for the association |(Rieii and .

R. opo 1989), and lamin white was used as a negative control. and others, 1993). Proteins, coded by p

SA 4-AO-constructions (roRTO), thank you! in a vertical column, and proteins coded by CA 4-A1 constructs

SAO-SN), tribute! horizontally. Two deletion mutants, marked with asterisks, were cloned by two-hybrid screening of the cDNA library of Ve cells. and (Siopiesp, Raiu A, Sa., O.5.A.) using p55-1C, cloned in rOVTO, as a "bait". In that screening, four of about 4xX109

Me, the cDNA clones tested were positive. However, it was found that three of these clones corresponded to cDNA regions for human p55-K (two identical clones encoding residues 328-426, and one

A clone encoding residues 277-426). The fourth clone was found to encode an unknown protein. oh Dannse about the expression of p-galactosidase! were obtained as average values from two independent analyses

GK) of transformants, and represented as the amount of p-galactosidase product (the unit of activity was determined as OPadgoh103/OPero yeast culture, and the reaction time is given in minutes. The limit of detection in this analysis was O0.0bed. Deviations between duplicate samples, in all cases, were less than 25905 from the average value (not determined). o Ip migo test for the interaction of the bacterial hybrid protein "p55-1C-glutathione-Z-transferase (551) with the hybrid protein "ro5-1sb-maltose-binding protein (MBRR) confirmed that robB- K is capable of self-association, and excluded the participation of yeast proteins in this binding (see above). The indicated self-association was not affected by either increasing the salt concentration or EOTA (see above). 60 To assess the functional role of the self-association of the "death domain", we investigated the mechanism by which the induced expression of rbo5-K or its fragments affects cells susceptible to

TME-cytotoxicity. The result of that analysis is shown in Fig. 7, which illustrates the ligand-independent stimulation of the cytocidal effect in the cells of Ne! a, transfected po5-K with its intracellular domain (po5-1C) or its fragments (including the "death domain"). 65 Fig. 7 schematically shows the readings! different DNA molecules, co-coordinating different types of TME receptors, included in the vector, which transfected Neg cells (from the left edge of Fig. 7); zxpression (left and middle column); as well as the viability (right column) of Neia cells expressing short-term different full-length proteins rbo5-K, p55-1C, or a fragment! p55-1C, or as a control, luciferase (OS) (each of them is shown on the left side! Fig. 7), however, expression was carried out using a tetracycline-controlled expression vector. Unshaded rectangles (in the left, middle, and right columns) in Fig. 7 correspond to cells transfected in the presence of tetracycline (μg/ml), which inhibits expression; and the shaded rectangles (in the left, middle, and right columns) in Fig. 7 correspond to cells transfected in the absence of tetracycline. TME receptor expression was assessed 20 hours after transfection using 7/0. EIzA, and an antibody against the extracellular domain of the receptor (see schematic illustration on the left side

Fig. 7), and by determining the binding of radioactively labeled TME with cells (middle column).

Cytocidal effect of transfected proteins was assessed 48 hours after transfection. The data are given for 1-3 experiments with similar quantitative results, where each design was used in duplicate. "MUO" - "did not determine".

Thus, it can be seen from Fig. 7 that when an expression vector is used, which carries out strictly controlled expression of transfected cDNAs with the help of a tetracycline-regulated transactivator (Sovvep and Vtsiaga, 1992), a simple increase in the expression of ro5-K in Her cells by means of short-term expression of transfected cDNA encoding a full-length receptor leads to massive cell death. When only one rbob5-1C was expressed, even higher cytotoxicity was observed. A significant level of cytotoxicity was also observed when only a part of p55-1C, containing mainly the "death domain" (residues 328-426), was expressed in cells of Ne a. On the other hand, expression of p55-1C fragments, which lack the "death domain" or contain only its part (or expression of the luciferase gene, used as an irrelevant control) did not have any effect on cell viability. The cytotoxicity of p55-1C was also confirmed using cells stably transformed with cDNA. These cells continued to grow when p55-1C expression was not induced, but when p55-1C was expressed, the cells died (see above). c) Other functions of the intracellular domain of the p5-TMP receptor

In order to determine whether other TMUE functions are stimulated by the self-association of the intracellular domain, which includes the "death domain", we analyzed the effect of increased expression of the full-length receptor (roo-K) and expression of the intracellular domain of the receptor (ro5-1C) on the transcription of interleukin-8 ( 11-8), which is known to be activated by TME (Maizizpita et al., 19881. i.

The result! therefore the study of representations! on Fig. 8, which illustrates ligand-independent (for inducing the expression of the 11-8 gene in Her a cells transfected with p55-K or p55-1C using a tetracycline-controlled construct (see also "General methods and materials, and Example 1) On panel A o

Fig. 8 illustrates the Northern blot analysis (see above, chapter "General methods and materials) of RNA (7 μg/lane) extracted from Her a (HT a-1) cells, untreated ("control") or treated (C TME") TME (500μ/ml, 4 hours), or NTia-1 cells 24 hours after transfection (in the presence or absence of tetracycline) cDNA for p55-1C ("p55-1C"), p55-K ("P5BB5 -K"), or luciferase ("OS"). On panel B

Fig. 8 illustrates Northern blot analysis, where 185-rRNA was stained with methylene blue in each of the samples shown in panel A of Fig. 8. shsh c Thus, as can be seen from Fig. 8, the transfection of Her cells! and the tetracycline-controlled construct, which contains po5-K cDNA, induced the transcription of 11 -8. Even stronger induction was observed in cells transfected with cDNA for ro5-1C. In both cases, induction occurred only in the absence of tetracycline in the growth medium, which indicates that the induction is the result of the expression of transfected ro5-K or ro5-1C Transfection of cells with luciferase cDNA, -I used as a control, did not have any effect on the transcription of the 11 -8 gene.

Based on the above results, it is obvious that a simple increase in the expression of p55-K, or even the expression of only its intracellular domain (rb55-1C) is sufficient for the induction of ligands

Te) (TME)-independent cytotoxicity and other effects, including increased expression of the 11-8 gene in 5p K cells. The induction of these effects, in all likelihood, is caused by the self-association of the intracellular o domain p5ob5-K (p55-IS). Since, as indicated above, it is obvious that after the self-association of rob-1C, the "death domain" is primarily responsible for signal transmission, it is possible that the induction of intracellular processes leading to the stimulation of cytotoxicity in cells, as well as other effects, for example, induction of 11-8 gene expression, mediated by other regions of p5bB-1C after their self-association. Therefore, it is quite possible that different regions of p55-1C are responsible for different

TME-induced effect! (for example, cytotoxicity and induction of 11-8 gene expression) in cells, iF) where the indication is effective! are stimulated in response to intracellular signal transmission after self-association with rob-16.

The fact that rb5b5-1C is capable of inducing ligand (TME)-independent stimulation of other intracellular effects, for example, inducing the expression of the 11-8 gene, means that p5o5-1C or its specific fragment! can be used as a highly specific tool for the implementation of the necessary effects in cells or tissues, without resorting, however, to the processing of those cells or TME tissues. In many pathological conditions (for example, malignant tumors), treatment of cells with tumor necrosis factor (TME), especially in high doses, can lead to undesirable side effects 65 Systemically induced by TME after its binding to its receptor. Thanks to the present invention, it was established that ro5-1C can imitate other specific TME-induced effects! (in addition to cytotoxicity), for example, induction of II/-8, which opens up the possibility of cell- or tissue-specific introduction of p5-1C or its specific fragments, which are capable of transmitting a signal to induce desired specific intracellular effects, for example, induction of I/-8, and the same makes it possible to avoid the occurrence of side effects, often observed in the process of TME processing. 4) Ligand-independent stimulation of cytotoxic effects in Neia cells, induced by intracellular domains and "death domains" p55-TME-K and EAB-K (HAB/AROT1)

As for the cytotoxic activity of the intracellular domains of TME-K and GA5B-K (roB51C and EGAZB-1C), 7/0, in accordance with the present invention, it was also found that p551cS, its "death domain" (pO5OO) and

EA5B-1C have the ability to ligand-independent stimulation of the cytocidal effect in Her cells! and. Therefore, Neia cells were transfected with expression vectors containing various constructs of either full-length p5-TME-K, its fragments, including p51C and p550pO, or EA5-1C. In one of the experiments, the cells were not cotransfected! constructions containing rob-TME-K u75 (roo-K) and RABY (in more detail, these constructions and their preparation are described above). The result! from the study of representations in Fig. 9 (A and B), where on the left side of panels A and B Fig. 9 schematically shows the construction, used for transfection of cells of Her a; in the two middle columns of those panels (second and third from the left) are graphical representations! TME or EA5 receptor expression results; and in the first part of the panels graphical representations! the results of research on the viability of transfected cells.

Fig. 3 shows the results for transfected NeHa cells expressing (with short-term expression) full-length rob5-K, ro5-1C or its fragment, or, as a control, luciferase (OS), and, in all cases, a tetracycline-controlled vector was used zxpressions On

Fig. 9 shows the results for transfected Neia cells expressing (with short-term expression) only EA5Z-1C or together with p55-K; and here is the result! were obtained with the use of a tetracycline-controlled expression vector. On the diagram shown in fig. 9A and

OB, unshaded rectangles correspond to cells transfected in the presence of i) tetracycline (1μg/ml), which inhibits expression, and shaded rectangles correspond to cells transfected in the absence of tetracycline. The expression of the TME receptor was evaluated 20 hours after transfection by EHIZA analysis using antibodies against the extracellular domain of the receptor (see the left panels), and by determining the binding of radioactively labeled TIME to cells (middle panels). The cytocidal effect of the transfected proteins was evaluated 48 hours after transfection. The data are from 1-3 experiments with similar quantitative results, where each construct was tested twice. "MO" means "not determined". From the results presented in Fig. 9A and 9B it is clear that at the expression of only one ro51C was observed even more)

З5 VsOoKaya cytotoxicity. A significant level of cytotoxicity was also observed when expressing only the "death domain" (ro5003). In contrast, expression of ro551cS fragments, which lacked the "death domain" or contained only its fragment, did not affect cell viability. Zxpression

EAB-1C did not lead to significant cytotoxicity, but, at the same time, it enhanced the cytotoxicity of co-expressed Rob5-K. "

Example C from c Other proteins that have the ability to bind to the intracellular domains of RBOB5-TME-K or EA5-K

And using the method described above in Example 1, three more were isolated and identified. a protein capable of binding to ro51C or EA5-1C.

Fig. 10-12 schematically shows a partial and preliminary nucleotide sequence

cDNA clones, designations Рg, РО, and ОО11, respectively. - Clones B2 and BE9 were isolated by screening the library of muscle embryos using muscle BAB-1C as a "bait". Fig. 10 schematically shows a partial nucleotide me) sequence from; E2 cDNA, which was sequenced. Fig. 11 schematically shows a partial one

Ge) nucleotide sequence of 1724 based on PO cDNA, which was sequenced. The analysis of the binding ability of proteins encoded by clones E2 and E9 (GE2 and E9, respectively) showed that: o a) E2 strongly interacts with human p5B51C and p5B5OYU and with muscle BA5B-1C, but, at the same time, it interacts weakly 4 ) interacts with irrelevant (control) proteins ZME1 and lamin, as well as with the relevant (i.e., considered in the present invention) human EAZ-1C protein;

B) GU strongly interacts with people. р55-1С and Мишинм ЕБА-1С but weakly interact with people. BA-1C (relevant protein) and irrelevant (i.e., not related to the present invention) proteins ZME1 and lamin;

F) c) None of E2 and E9 engages with people. p751C, rovt9 ("empty" vector - "bait") or chel. CO-40. In addition, studies of data banks for genes and proteins ("SepeVapk" and "RgsivipVapk") showed that E2 and E9U are new proteins. Thus, E2 and E9 are new proteins that specifically bind to EA5Z-1C and p551cC.

Clone 0011 was isolated by screening No! human a-libraries with use as a "bait" of ro5OYU. Fig. 12 schematically shows a partial nucleotide sequence of 425 bases of OO11 cDNA, which was sequenced.

Clone 0011 has a length of approximately 800 nucleotides. The full-length transcript, the length of which is 65 C, is approximately 12 kCr, and was probed using this clone. Analysis of the binding ability of the protein encoded by clone 0011 showed that 0011 strongly interacts with robo

(amino acid! 326-414) (see Fig. 9) and does not interact with deletion mutants of that domain, for example, with "a.k. 326-404". 0011 also works with people and people. EAB-1C, and to some extent with lamin.

However, 0011 does not interact with 5ME1, nor with roVvte ("empty" decoy vector"). In addition, it was not found in any of the data banks ("Zepe Vapk" and "Rgsiveip Vapk"). Thus OO11 is a protein that specifically binds to ro51cS (ro5100)) and GA5-1C.

Example 4

Construction of soluble TA7E-dimeric receptors

Based on the data obtained above in Example 2, on the basis of which it was established that 70 intracellular domain p55-K (ro55-1C) and its part ("death domain"), as well as the intracellular domain GAB/APO1 and its part (also called the "death domain"), similar to the "death domain" p55-1C, have the ability to self-associate, it became obvious that it is possible to construct a new TME-receptor!, which is capable of self-association (aggregation), and which is recognized as soluble. Hybrid proteins containing, basically, the entire extracellular domain of p5b-K, compounds, mainly with the complete 7/5 Intracellular domain or the "death domain" of p5-K or RAB/AROT, can bind to such receptors. Such structures do not contain the transmembrane domain of rb55-K (HAB/AROT), and therefore they are soluble. In addition, due to the ability to self-associate intracellular domains or their "death domains", these hybrid structures will have the ability to oligomerize with the formation of at least dimers (and possibly multimers of a higher order) of ro5-K. Accordingly, such dimeric TME-receptors (roB5-K) will be capable! bind to at least two monomers of the natural TME homotrimer, thereby forming a soluble TME receptor that binds with a high degree of avidity to its ligand (homotrimeric TME).

Based on the above, four types of p55-TME-receptor hybrid proteins can be constructed, each of which has the ability to oligomerize and is soluble: c) Hybrid product formed between the extracellular domain of p5b5-K (EC55) and the intracellular domain of p5b5-K (work5-1C); i) i) Hybrid product formed between EC55 and "death domain" p55-1C0 C(0055); ii) Hybrid product formed between EC55 and the Rabz/AROY intracellular domain (1SEAZB); and 1M) Hybrid product formed between EC55 and "death domain" ISEAZ (PERSON). so z In each of the above-mentioned hybrid proteins, the ability to bind to a monomer. TME is provided by the EC55 part of the protein, while oligomerization (or at least dimerization) of each of the specified types of proteins is provided by its "tail" part, which is any of the regions of ro51C, 0055, b1CEAS0, or PERSON.

For the construction of more specific hybrid proteins, the standard technique of recombinant DNA, which is widely used by specialists at the present time, can be used. for example, cha

ZatbrgooK et al., (1989) MoiIesshag Siopipd A Iarogaigu Mapia!i, Soii Zrgipd Nagrog I arogaigu Rgevzv, Soii

Zrgipd Nagrog, M. M. For these purposes, any suitable expression vector (cloning system or plasmid intended for expression of selected inserted DNA) can be used, which originates from bacteria, bacteriophages, or animal viruses, and in which it can be introduced, in "one or more stages, DNA encoding EC55 and one of the "tails" which are ro5-1C, 0O55, 1SEAZ or from c OOGAZ. DNA inserted in this way, encoding each of the hybrid proteins, can be placed

And under the control of various regulatory sequences of systems! cloning or plasmids, for example, and such as promoters, the site! connection with the ribosome, site! binding of transcription factors, etc. The choice of such regulatory zxpression sequences depends on the type of the selected zxpressing vector, and usually on the type of host cell (zukaryotic or prokaryotic), in which it is desirable to express the hybrid proteins of the present invention. Preferable host cells (and consequently, expressing vectors) are zukaryotic cells, and in particular, cells

Me. mammals A DNA molecule encoding each of the above hybrid proteins can be obtained and inserted into an expression vector using the following procedures: a) First, using standard techniques, a series of oligonucleotides is constructed for use in the polymerase chain reaction, where the indicated oligonucleotides are: 73 es tu os sto os so be: SHEEBO. KANVI" BO o V yu M. Zhebsh sheE sl NVO Kuby ov b U OTO Ebbya" VZHY" etc.

Z KOBe SheS AYU BO Z OM, SHO OE VAS FKOBe; BESHCHEVF" and nem ns dos s but NK no, NN VINY EE en SHY dela Love r; B bresivvayetyah IY sii b5 le 2 x y slovana nya sekini rikakh Zieyu E I

Е AV ve: в и НЕ НН ОВ nazhretiyuyyuchotvtkwo V sezhne este and RO st tk vdo se orevh her

Shchy abso tr to SYA MU ZHAE YANG SHO yah o vovyh

B) Plasmids containing clones of the full-sized p55-K and RGAB/APO1 receptor, and obtained in our laboratory (see also simultaneously considered application ERB568925I and Example! 1-3 of this application), are subjected to the following manipulations to obtain DNA fragments, which encodes each of the hybrid proteins, and which are then ligated into the above-selected expressing vector: i) To obtain a DNA fragment encoding EC55, which is a component of all four c hybrid proteins, RSE is performed. on a plasmid carrying human cDNA. p55, using the above-mentioned He) oligonucleotides MoMo1 and 2 (the size of the fragment is 640 p.0.). i) To obtain a hybrid product EC55-1C55, perform RSK on a plasmid carrying human cDNA. p5B5, using oligonucleotide MoMoZ and 4, as a result of which a DNA fragment encoding 1C55 (677 p.0 in size) is obtained, which is then mixed with EC55, a hydrolyzing enzyme, and ligated at blunt ends into any expression vector under control of the corresponding promoter. The orientation of the EC55-1C55 sequence inserted into the vector is checked by restriction hydrolysis and by sequencing. Me. her) To obtain the hybrid product EC55-1C RA, 1TSEA5 is produced with the help of PCR on a plasmid carrying the cDNA for RAB, using oligonucleotide MoMo5 and 6, as a result of which a fragment 3o (size 448 p.0.) is obtained, which is then cut with an enzyme They are mixed with EC55, cleaved with an enzyme - ZAI, after which they are ligated at the blunt ends into the expressing vector under the control of the appropriate promoter. The orientation of the inserted EC55-1SEA5 in the vector is checked by restriction hydrolysis and sequencing. "im) To obtain the hybrid product EC55-0O055, produce a DNA fragment containing the rBBb sequence using PCR on the cDNA encoding human p55, and using c) oligonucleotide MoMo7 and 4. The resulting product is 314 bp. they are cut with zZaII enzyme and mixed with z»ES55, cut by ОО11, after which they are ligated at the blunt ends into the mammalian expressing vector.

The orientation of the inserted product EC55-00О055 in the vector is verified by restriction hydrolysis and sequencing. m) To obtain the ES55-00 RAB hybrid product, produce a DNA fragment that co-iodines ЮОБА5 with the help of RSK on the RAB cDNA using the oligonucleotide MoMob and 8. The resulting product is

F 332p.o. they are cut with the enzyme zZaII and mixed with EC55, hydrolyzed zZaY, after which they are ligated at the blunt ends into the mammalian expression vector. The orientation of the inserted EC55-OOBAZ product in the vector is verified by restriction hydrolysis and sequencing. 2) 20 After the construction of the above-described expression vectors, they can be introduced, for the purpose of their expression, into suitable mammalian cells (for example, Chinese hamster ovary cells (CHO) or monkey kidney cells (CO5)) using standard methods. Hybrid proteins expressed in this way can be purified! by standard methods | see, at the same time we are considering application EP 308378; ER 398327; and ER 568925). Purification of hybrid proteins can be performed, then analyzed for their ability to oligomerize (and the degree of such oligomerization, i.e., the ability of proteins to form

GF) dimers or multimers! higher order), as well as their ability to bind to TME (and the affinity or avidity of such binding). and Example 5

Construction of soluble GAB/APO1 dimeric receptors 60 By a method similar to the method described in Example 4, many products can be produced! four types of hybrid RGAB/APO1 proteins, each of which has the ability to oligomerize and is soluble; namely: i) The hybrid product formed between the extracellular domain of RAB/AROT1 (ES GA5) and the intracellular domain of ro55-1C; b5 her) Hybrid product, formed between EC GAZ and "domain of death" rb55-1C (0055);

ii) Hybrid product formed between ES RAZ and the intracellular domain of RAB/APO1 (1C ГА5Б); and them) A hybrid product formed between EC RAZ and the "domain of death" 1C GAZ (OOGASB).

In each of the above-mentioned hybrid proteins, the ability to bind to the PHA5 ligand is ensured

EC ERAz is a part of the protein, while oligomerization (or at least dimerization) of each of the specified types of hybrid proteins is provided by its "tail" part, which is any of the p55-1C, 0O55, 1C EAB5, or OOBAZ regions.

Constructions of DNA fragments encoding multiple hybrid proteins and an expression vector containing these constructions can be obtained as described in Example 4, except that at 7/0 Zztom, dolzhn! other suitable oligonucleotides (not shown) can be used, after which these constructions can be used to obtain a fragment of EC RAB intended for ligation with any of the above-mentioned "tail" parts. After that, expressing the vector! you can enter! in a suitable host cell, and the hybrid proteins obtained as a result of expression can be purified! and analyze! on their ability to oligomerize (and the degree of such oligomerization, i.e., on the ability of these proteins to form dimers or multimers of a higher order), as well as on their ability to bind to the HA5 ligand (and the affinity or avidity of such binding).

Example 6

Construction of soluble oligomeric "mixed" TME/RAB5 receptors

In order to obtain oligomeric receptors with "mixed" affinity, i.e., affinity for two ligands, TME and RA5-K-ligand, the above-mentioned hybrid proteins (Examples 4 and 5) can be used in the following procedures: i) obtaining a hybrid product , described in Example 4, and containing the extracellular domain of TME-K (p75

TME or p55-TME-K), compounds with one of: p5-1C, EAB-1C, p5OO, or RABO; i) obtaining the hybrid product described in Example 5, which contains the extracellular domain of EAZ-K, and compounds with one of: po51C, RA5B-1C, ro5OO, or EAB-OO; and ii) mixing any one of the hybrid products (ii) with any one of the hybrid products (ii) for i) obtaining a new dimeric (or higher order oligomeric) receptor, which contains both TME-K and GA5-K extracellular domains, the compound by means of their -1С or -ЯО regions.

In the above procedures, a hybrid product! (ii) and (ii) are able to receive! separately, namely, as a result of their elimination from the transformed cells in which they were produced, and then, these products can be mixed and mixed with the formation of receptors with "mixed" affinity. at

Alternatively, host cells can be transfected with vectors carrying sequences encoding hybrid proteins of both types; and, in that case, a receptor! with "mixed" affinity can be obtained directly from co-transfected cells. In fact, oligomerization of hybrid products with the formation of oligomeric receptors can occur in cells during or after the procedures! cleaning, as a result of which, you may suffer! hybrid product!, expressed in cells. For the specific selection of receptors with "mixed" affinity, any known method can be used, for example, affinity chromatography, in which to detect receptors with extracellular domains! of both types, antibodies against TME-K and EA5-K extracellular domains are used in successive chromatographic stages. with c SsIlki: . Adegka, O., EpdIietapp, N., Notik, M., ZkoppiskK, M., Yemo, U., MaPasy, O. apa Kizpiai, 0. (1991) Sapseg Kev. I! and? 5602-5607.

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Claims (14)

The formula of the invention 1. An isolated DNA molecule encoding a polypeptide capable of binding to the intracellular domain of the po5-TME receptor, including: (a) a DNA sequence encoding a polypeptide with residues 202-426 of the po5-TME-K amino acid sequence presented in ZEO IU MO:25 , or (B) a DNA sequence encoding a fragment of a polypeptide encoded by DNA specified in subparagraph (a), where the specified fragment preserves the ability of the polypeptide of which it is a fragment to bind to the intracellular domain of rob5-TME-K, where the sequence of the specified DNA molecules ! encodes the sequence represented by ZEO IYU MO:25 and consisting of no more than residues 202-426 of 5BEO IYU MO:25. 2. An isolated DNA molecule according to claim 1, encoding a protein selected from the group consisting of proteins designated as 55.1 and 55.3. Z. Isolated DNA molecule according to claim 2, encoding protein 55.1, having an amino acid (o) sequence corresponding to amino acid residues 328-426 of the amino acid sequence ZEO IU MO:25. 4. An isolated DNA molecule according to claim 2, encoding protein 55.3, having an amino acid sequence corresponding to amino acid residues 277-426 of the amino acid sequence ZEO IU MO:25. co 5. Vector containing the DNA sequence according to claim 1. o 6. Transformed host cell containing the vector according to claim 5. 7. A method of producing a protein, its analogue or derivative capable of binding to the intracellular 335 domain of the rob-TME receptor, which provides for the cultivation of transformed host cells according to claim 6 under conditions suitable for the expression of the specified protein, its analogue or derivative; implementation of post-translational modifications of the specified protein, if necessary to obtain the specified protein; and extraction of the specified expressed protein, its analogue or derivative from the media! cultivation of the specified transformed cells or from cell extracts of the specified transformed cells. "20 8. A protein, its analogue or derivative, encoded by the DNA sequence according to claim 1, and the protein, its analogue or derivative is capable of! bind to the intracellular domain of rob5-TME-K. 9. Protein according to claim 8, selected from the group including proteins 55.1, 55.3 and 55.11, and their biologically active analogs and derivatives. 10. The protein according to claim 9, including the amino acid sequence of protein 55.11. 11. Antibody having specificity in relation to the protein, its analogues or derivatives according to claim 8. -I 12. A pharmaceutical composition for modulating the action of the p55-TME-K ligand on cells, containing as an active ingredient the protein according to claim 8 or the protein rb5bis or p5OrO, their biologically active fragment, Me, analogues, derivatives or their mixtures. co 13. A pharmaceutical composition for modulating the action of the p55-TME-K ligand on cells, containing as an active ingredient a recombinant, derived from an animal virus, a vector encoding a protein capable of binding to a cell surface receptor, and encoding a protein according to claim 8 or a protein c» robBIS or ro5O0, their biologically active fragment or analogues. 14. Pharmaceutical composition according to claim 12 for processing cells by inducing TME-associated effects in them, containing as an active ingredient robiC, its fragment, an analogue of mly derivative, and a pharmaceutically acceptable carrier. F) name) 60 b5