Polypeptide possessing capacity for binding with intracellular domain p-55 of tnf-receptor, dna molecule encoding this polypeptide, expression vector and method for preparing polypeptide

FIELD: molecular biology, genetic engineering, polypeptides, medicine.

SUBSTANCE: in using the double-hybrid yeast system DNA sequences encoding polypeptides (55.1 and 55.3) have been found that elicit ability for binding with intracellular domain p-55 (p-55IC) of TNF-receptor. It has been established that these polypeptides represent fragments of amino acid sequences p-55IC, respectively, from 338 to 426 and from 277 to 426 residues. As result of insertion of DNA fragments with a sequence encoding polypeptide 55.1 or 55.3 into the structure of expressing vector and transformation suitable host-cells by this vector recombinant form of indicated polypeptides have been prepared. Using this invention provides the possibility for modulating the function of intact p-55 of TNF-receptor. Invention can be used in medicine in treatment of diseases associated with transfer of TNF-signal.

EFFECT: improved preparing method and valuable properties of polypeptide.

9 cl, 17 dwg, 3 tbl, 6 ex

 

The present invention relates to receptors belonging to the superfamily of receptors of the TNF/NGF, and the regulation of their biological functions. To the superfamily of receptors of the TNF/NGF receptors belong growth factors tumor necrosis P55 and P75 (TNF-R), the receptor for FAS ligand (also called FAS/AP01 or FAS-R, and denoted hereinafter FAS-R) and other receptors. More specifically, the present invention relates to novel proteins that bind to the intracellular domains (1C) P55 and p75-TNF-R and FAS-R (denoted hereinafter RS, RS and FAS-1C, respectively), and which have the ability to modulate the function of the P55 and P75 TNF receptor and FAS-receptor. One of these proteins, the ability to communicate with RS intact p55-TNF-R, is itself RS in the form of molecules hrs or part thereof, for example, so-called "domain of death" RS. Thus, the present invention also relates to new TMF-associated effects that can be induced in cells by intracellular domain of the p55-TNF (RS) or part of the ligand (TNF)-independent manner. In addition, the present invention relates to the production and use of these new proteins that bind to the P55 and p75-TNF-R, and proteins that bind to the FAS-R, which in this description referred to as RS-, RS - and FAS-1C-binding proteins.

In another aspect, the present invention also relates to new plants is oronym oligomeric TNF-R, oligomeric FAS-R and oligomeric receptors, representing a mixture of TNF-R and FAS-R; to the use of these receptors, and to methods for their preparation.

Background of invention

The tumor necrosis factor (THF-α) and lymphotoxin (TNF-β) (also called TNF-α and TNF-β, respectively) are multi-functional cytokines produced mainly by mononuclear phagocytes, and has multiple effects on the cell (Wallach, D. (1986), "Interferon 7 (Ion Gresser, eo.), pp.83-122, Academic Press, London; and Beutler & Cerami (1987)). The action of both of these cytokines, TNF-α and TNF-β initiated by their binding to specific receptors on the cell surface. Obviously, some of these cytokines have a positive effect on the body, that is, they may, for example, to destroy a tumor or virus-infected cells and to potentiate the antibacterial activity of granulocytes. In this case, TNF protect the body against tumors and infectious agents, as well as promotes the healing of wounds. Thus, TNF can be used as an antitumor agent that binds with its receptors on the surface of tumor cells and thereby initiates the events leading to the deaths of these tumor cells. TNF can also be used as anti-infective means the A.

However, both these factors, i.e. TNF-α and TNF-β, also have adverse effects. It was shown that overproduction TNF-α may play an important role in the pathogenesis of some diseases. For example, currently it is already known that the action of TNF-αmainly on the vascular network of the body, causing symptoms of septic shock (Tracey et al., 1986). In some diseases, TNF can also cause sudden weight loss (cachexia) due to suppression of the activity of adipocytes and provoking anorexia, and therefore the tumor necrosis factor TNF-α is also the name of cachexin. It was also reported that TNF mediates tissue destruction in rheumatoid diseases (Beutler & Cerami, 1987) and is the main mediator of rejection observed in the reactions of "graft versus host disease" (Piquet and others 1987). In addition, it is known that TNF is involved in inflammation and mediates many other diseases.

Two different from each other and independently expressed receptor P55 and p75-TNF-R, which are specifically associated with TNF-α and TNF-β initiate and/or mediate the biological functions of TNF above. These two receptors are structurally distinct intracellular domains, suggesting that they transmit different signals (see, Hohnman and others, 1989; Engelmann and others, 1990; Brockhaus and others, 1990; Leotscher, etc, 1990; Schall and others, 1990; Nophar and others, 1990; Smith and others, 1990; Heller and others, 1990). However, cellular mechanisms, such as various proteins and possibly other factors involved in the transmission of intracellular signal P55 and P75 TNF-R, not yet identified (below first describes the new proteins are able to bind with RS and RS). But it is this transmission of signals, which usually occurs after binding of the ligand (i.e. TNF-α or TSR-β) receptor, responsible for initiating the cascade of reactions that eventually lead to the observed cellular response to TNF.

With regards to the above Titorenko actions of TNF, in most cells studied to date, this action is stimulated mainly by receptor p55-TNF. Antibodies against the extracellular domain (the domain to bind to the ligand) P55-TNF-R, may themselves be stimulants Titorenko effect (see, EP 412486), which correlates with the efficiency of cross-linking of the receptor with antibodies, and which is, obviously, the first stage of the transfer process of the intracellular signal. In addition, research conducted by the method of mutations (Brackebuch and others, 1992, Tartaglia and others, 1993), showed that the biological function of the p55-TNF-R depends on the integrity of its extracellular domain, which suggests that the initiation of intracellular signal transduction, induc the dominant cytocine the action of TNF, is the result of combining two or more intracellular domains of the P55 TNF-R. in Addition, TNF (α and β) Fightstar in the form of homotrimer, and it has been suggested that induces p55-TNF-R-mediated transmission of intracellular signals due to its ability to bind and cross-linking with receptor molecules, i.e. to the creation of the aggregation of the receptor. Below, in the present application will be described how RS and 55DD may be associated with each other, and to induce ligand-independent manner, TNF-associated effects in the cells.

Another member of the superfamily of TNF/NGF receptor is FAS receptor (FAS-R), which is also called FAS-antigen, which is a protein found on the cell surface expressed in various tissues, and has homogay with a number of cell surface receptors, including TNF-R, NGF-R, FAS-R mediates cell death by type of apoptosis (Iton. and others, 1991), and is obviously, as a negative selector self-reactive T cells, i.e. in the process of maturation of T cells, FAS-R mediates apoptosis of T cells that recognize autoantigens. It was also found that mutations in the gene FAS-R (1 pr) cause a violation of lymphoproliferation in mice that have human-like picture of such autoimmune diseases as systemic lupus erythematosus (SLE) (Watanabe-Fukunaga and D. the., 1992). The ligand for FAS-R is, obviously, a molecule associated with the cell surface, and carried, among others, T-killer cells (or cytotoxic T-lymphocytes CTLs), and therefore, when such CTL contact with cells bearing FAS-R, they are able to induce apoptosis of FAS-R-bearing cells. Furthermore, it was obtained a monoclonal antibody with specificity to FAS-R, which had the ability to induce apoptosis of cells carrying FAS-R, including mouse cells transformed with cDNA that encodes FAS-R man (Iton and others, 1991).

It was also discovered that in addition to T-lymphocytes, there are other normal cells that Express FAS-R on their surface, and which can be destroyed by stimulation of this receptor. It has been suggested that unregulated induction of this process of cytolysis leads, in some diseases, the destruction of body tissues, for example, to destruction of liver cells in acute hepatitis. Therefore, the development of ways to reduce the cytotoxic activity of FAS-R may lead to therapies that are important.

And Vice versa, because it was also found that some cancer cells and HIV-infected cells are FAS-R on their surface, antibodies against FAS-R or FAS-R ligand can be used to stimulate FS-R-mediated cytotoxic effects, that will allow you to obtain the means of destruction specified malignant or HIV-infected cells (see, Iton. and others, 1991). In addition, for other ways to enhance the cytotoxic activity of FAS-R may also have important therapeutic value.

Long overdue a need for methods of modulating the cellular response to TNF (α or β) FAS-R ligand. For example, in pathological processes mentioned above, where surgentes TNF or FAS-R ligand, it is desirable to inhibit TNF - or PAS-R-ligand-induced cytosine effects; and in other situations, for example during wound healing, it is desirable to stimulate the action of TNF, or, in the case of tumor or HIV-infected cells, it is desirable to stimulate FAS-R-mediated response.

The authors of the present invention was developed several ways (see, for example, the application for the European patent EP 186833, EP 308078, EP 398327 and EP 412486) regulation undesirable effects of TNF by inhibiting the binding of TNF to its receptors using antibodies against TNF or by using soluble TNF-receptors (which is, basically, soluble extracellular domains of the receptors) to compete for binding of TNF with TNF receptor associated with the cell surface. In addition, based on the fact that to receive TNF-induced cleoc the CSO must answer the TNF binding with their receptors, the authors of the present invention have been developed (see, for example, EPO 568925) regulate activity of TNF by modulating the activity of TNF-receptors. In particular, in EPO No. 568925 described by way of modulation of signal transduction and/or hydrolysis in TNF-R, resulting in peptides or other molecules can interact either with the receptor or effector proteins that interact with the receptor, thereby making the modulation of the normal functioning of the TNF-R. EPO 568925 also describes the design and characterization of various mutant P55 TNF receptors with mutations in the extracellular, transmembrane and intracellular domains of the p55-TNF-R. Thus, the region located above the domains of the P55 TNF-R were identified as major areas of functioning of the receptor (i.e. binding ligand TNF) and subsequent signal transduction and intracellular signaling, leading, ultimately, to TNF-effect observed on the cells. In addition, this patent describes several methods for the isolation and identification of proteins, peptides and other factors that are able to communicate with different areas in the above domains of TNF-R, and these proteins, peptides and other factors may be involved in the regulation or modulation of the activity of TNF-R. EPO 568925 also revealed how the selection and is klonirovania DNA sequences, encoding these proteins and peptides; methods of constructing expressing vectors for producing these proteins and peptides; and methods of obtaining antibodies or their fragments that interact with TNF receptors or with the above-mentioned proteins and peptides that bind with different areas of the TNF-R. However, in EPO 568925 there are no descriptions of proteins and peptides of the present invention that are associated with the intracellular domain of TNF-R (e.g., p55-TNF-R), a also lacks any description of the isolation and identification of such proteins or peptides using yeast twohybrid system. In addition, still have not been described proteins or peptides are able to bind with the intracellular domain of FAS-R.

Thus, for inhibiting the action of TNF or FAS-R ligand is necessary to reduce the number or activity of TNF-R or FAS-R on the cell surface, whereas stimulation of the actions of TNF or FAS-R ligand is necessary to increase the number or activity of TNF-R or FAS-R For this purpose recently, the authors of the present invention were sequenced and analyzed the promoters of the P55 TNF-R and p75-TNF-R, in the result, it was found that a number of their key "motifs" are sequence-specic factors in the regulation of transcription; and therefore, in essence, the expression of TNF-R may re wirematic at the level of their promoter, i.e. can be achieved by inhibition of transcription initiated by the promoter, in order to reduce the number of receptors or enhancing transcription initiated by the promoter in order to increase the number of receptors (see, 1L 104355 and 1L 109633, and their respective, not yet published European patent and the PCT-patent). Of relevant studies relating to the regulation of FAS-R at the level of promoter FAS-R gene, not yet reported.

In addition, it should also be noted that although, as you know, the receptors of the tumor necrosis factor (TNF) and their structurally related receptor FAS-R (stimulator cells)after stimulation of leukocyte-produced ligands, are destructive activity that leads to their own destruction, however, the mechanisms of this stimulation are still obscure. Mutational studies have shown that FAS-R and P55-TNF receptor (P55-R)input signals cytotoxicity, have in their intracellular domains specific areas (Brackebush and others 1992; Tartaglia and others, 1993; Iton and Nagata, 1993). These areas (the"domain of death") are similar in sequence. Such "death domains" of both FAS-R and P55-R tend to self. The self-Association of these domains, it is obvious that stimulates the aggregation of these receptors, which is required for the initiation of signal transmission (as shown below and as described in the Song and the RV, 1994; Wallach and others; 1994; Boldin and others, 1995), and at high levels of expression of the receptor can lead to stimulation of ligand-independent signaling (as shown below and as described Boldin and others, 1995).

Thus, until the present invention has not been any reports of proteins that can regulate the action of ligands belonging to the superfamily of TNF/NGF (for example, the effect of TNF or FAS-R ligand on cells), Uporaba the transmission of the intracellular signal that is controlled, in all probability, the intracellular domains (1C) receptors belonging to the superfamily of receptors of the TNF/NGF (such as TNF-receptors), namely the intracellular domains of the P55 and p75-TNF-R (p551C and RS, respectively), and FAS-1C.

In line with this, one of the purposes of the present invention to provide a protein having the ability to bind to the intracellular domain of TNF-R and FAS-R, and these proteins, as currently anticipated, involved in the intracellular signal initiated by binding of TNF to its receptors, or binding of FAS-ligand to its receptors.

Another objective of the present invention is to provide antagonists (e.g. antibodies) against these proteins bind to the intracellular domain (1C-binding antibody), which, if necessary, can be used is carried out for the inhibition of the transmission signal, in that case, if such 1C-binding proteins are positive effectors of signal (i.e. induce a signal transmission), or they can be used to amplify the transmission signal, if such 1C-binding proteins are negative effectors of signal (i.e., inhibit signal transmission).

Another objective of the present invention is the use of such 1C-binding proteins for isolation and characterization of other proteins or factors, which may, for example, to participate in further processes the signal, and/or to highlight and identify other receptors involved in the previous process of signal transmission, with which contact these 1C-binding proteins (for example, other TNF-R or related receptors), and therefore, for functions which these proteins are also responsible.

In addition, the present invention is the use of the above 1C-binding proteins as antigens for the production of polyclonal and/or monoclonal antibodies against these proteins. Thus obtained antibody can be, in turn, used to clean new 1C-binding proteins from various sources, such as cell extracts or transformed cell lines.

These antibodies can also be used in diagnostic the fir-trees, for example, to identify disorders associated with impaired cellular functions mediated by receptors belonging to the superfamily of TNF receptors/NCP.

Another objective of the present invention to provide pharmaceutical compositions containing the above 1C-binding proteins, and pharmaceutical compositions containing antagonists 1C-binding proteins, for the treatment or prevention of TNF-induced or FAS-ligand-induced States; moreover, these compositions can be used to enhance the action of TNF or FAS-ligand, or for inhibiting the action of TNF or FAS-ligand, depending on the nature of the above 1C-binding protein or its antagonist contained in this composition.

In addition, in accordance with another objective of the present invention, the present description of the disclosed other methods of elimination or inhibition of endogenously generated or exogenously introduced TNF or FAS-R ligand through the use of soluble oligomeric TNF-receptors, oligomeric FAS-receptor, or oligomers, representing a mixture of TNF receptor and FAS receptors. In this regard, it should be noted that was made one attempt to isolate and produce using the technique of recombinant DNA TNF-binding protein, called TBP-1, which, as was shown, the possession is the ability to inhibit the action of TNF. Antagonism of the indicated protein was determined by measuring the reduction of the cytotoxic activity of TNF, as well as by measuring the level of inhibition of TNF binding to their receptors (ER 308378). As it was found that TBP-1 protects cells from the toxic effects of TNF at concentrations of several nanograms per one milliliter and prevents the binding of TNF-α and TNF-β cells with the simultaneous introduction of this protein with the indicated cytokines. A subsequent study of the mechanism by which acts TBP-1, revealed that TBP-1 does not interact with the cell-target, but rather blocks the function of TNF by specific binding to TNF, competing, thereby, to its receptor.

Later, using a different cleaning method was found fightstyle two active ingredients, one of which is TBP-1, and the other is the second TNF-binding protein, called TBP-11 (first described in EP 398327). Both of these proteins protect cells from Titorenko in vitro action of TNF, and both proteins was associated with TNF-β less effective than TNF-α. Although the analysis carried out by electrophoresis in SDS page with LTOs, it was found that proteins TBP-1 and TBP-11 have very similar molecular weight, however, they can be distinguished from each other by the lack of immunological cross-reactivity,the difference N-terminal amino acid sequence and the difference in amino acid composition.

However, the above-mentioned TNF-binding proteins are Monomeric and the ability to communicate with only one monomer homotrimer TNF, a natural ligand, resulting in this TNF is still active (i.e. not fully neutralized) due to the fact that this trimer has two active monomer is not associated TNF-binding proteins. In addition, still have not been described soluble FAS-R (soluble proteins that bind to the FAS-R ligand)that is capable of contacting the ligand for FAS-R, which, as you know, is homotrimeric molecule associated with the cell surface.

The so-called "death domain (cells)" receptor P55 TNF (indicated by the P55-1C) was described in Tartaglia and others (1993), however, this work is not shown (as is done in this application)that the P55-1C and its "domain of death" are samoassotsiiruyutsya, and this self-Association is responsible, mainly, for signal transmission, resulting in the induction of cytotoxic effects on the cell. In addition, this publication is not reported in the possibility of producing a soluble, oligomeric TNF-receptoras or soluble oligomeric FAS-receptor, and also silent about the other TNF-associated effects induced P55-1C or its fragments, for example, about the effects of inducing gene expression of L-8, which are considered in the present invention. In another paper, published after the filing of the present application, discloses the ability to aggregation (i.e. self) P55-1C, but also does not mention anything about either obtain soluble oligomeric TNF receptor and FAS-receptor or other TNF-associated effects induced ligand-independent way P55-1C or fragments disclosed in this application.

Brief description of the invention

When working on the present invention we have discovered a new protein that has the ability to communicate with the intracellular domain of the P55 TNF receptor (RS-binding proteins), P75-TNF receptor (RS-binding proteins), and FAS receptor (FAS-1C-binding proteins). These RS, RS and FAS-1C-binding proteins can act as mediators or modulators of effector action of TNF or FAS-R ligand on cells by mediation or modulation of intracellular signal transduction, which occurs after the binding of TNF to P55 and/or P75 TNF-R or binding of FAS-R ligand on the cell surface. In addition, it was unexpectedly discovered that RS and FAS-1C have the ability to self, and that the fragments RS and FAS-1C are equally capable of contact with the P55-1C, especially with the so-called "death domains (cells)" (DD)in the intracellular domain (1C) of these receptors, i.e. 55DD and FAS-DD. So about what atom, the P55-1C and FAS-1C and fragments thereof also are proteins that are able to communicate with RS and FAS-1C, and therefore they can be modulators of the action of TNF or FAS-R ligand on the cell.

In addition, in accordance with the present invention has been more fully disclosed the nature of the binding of one of the new proteins (indicated in the present description protein 55.11) with the intracellular domain of the P55 TNF receptor (see Example 1).

Moreover, in another aspect, the present invention is based on the discovery of the fact that the intracellular domain of the receptor p55-TNF (P55-1C); the area contained therein (so-called "death domain" of the P55-1C); and the intracellular domain of the receptor and FAS/APO1 (FAS-1C); and the area contained in it (the so-called "death domain" FAS-1C), have the ability to self. Accordingly, it is possible, using standard techniques of recombinant DNA construct of soluble oligomeric TNF-receptor, which is a hybrid product that contains, at one end, at least two extracellular domain of the TNF-receptor, and the other of its end, at least two of the above samoassotsiiruyutsya intracellular domain, or parts thereof, where as a result of this self-formed oligomer having at least two hybrid product connected together. Thus, the Rast is orily oligomeric TNF-receptor able to bind with the two monomers natural homotrimer TNF, and as such effectively neutralizes the activity of TNF. Neutralization of TNF activity is desirable in all of the above conditions, where there is produced an endogenous or introduced exogenous excessive amount of TNF, leading to unwanted side effects. In addition, the effective binding with TNF soluble oligomeric receptors of the present invention may also serve to bind exogenously added TNF and its subsequent desirable prolongirovannogo release into the conditions under which the introduction of TNF has a beneficial effect, for example in tumour therapy. Similarly, using standard techniques of recombinant DNA, can be constructed oligomeric FAS-receptor, which is a hybrid product that contains at least two extracellular domain of the FAS-receptor on one end, and at least two of the above samoassotsiiruyutsya intracellular domain or its fragments, where as a result of this self-formed oligomer having at least two hybrid product connected together. Thus, the oligomeric FAS-R is able to contact the two monomers natural homotrimer FAS-R ligand, and effectively neutralize the activity of the FAS-R ligand. Neutralization activity of the FAS-R ligand is one which SJ is desirable in all the above situations, where an excessive amount of this ligand is associated with undesirable side effects. Similarly, if we take into account recent reports indicate a possible Association between TNF and FAS-R ligand-induced effects on cells and, consequently, on their geographical Association at the cell surface, where they bind with their receptors, it is obvious that using the standard techniques of recombinant DNA can be constructed mixed oligomeric receptor with specificity as to the TNF and FAS-R ligand. This mixed oligomer should be a mixture of the above hybrid products containing at least one extracellular domain of the TNF receptor and at least one extracellular domain of the FAS-receptor at its one end, and at least two of the above samoassotsiiruyutsya intracellular domain, or parts thereof at its other end, where as a result of this self-formed mixed oligomer having at least two such hybrid product, United together. Thus, the mixed oligomer capable to contact at the same time, at least one monomer of TNF and with one monomer FAS-R ligand, thereby, reduce or effectively neutralizing activity and TNF and FAS-R ligand nor the cell surface under conditions when, as stated above, excessive amounts of these two cytokines are associated with adverse cellular effects. As mentioned above, FAS-R ligand, mainly associated with the cell surface, and in recent publications also described forms of TNF associated with the cell surface. So the mixed oligomers TNF/FAS-R are particularly effective for neutralizing the activity of TNF and FAS-R ligand on the cell surface.

In accordance with this present invention relates to a DNA sequence that encodes a protein that is able to communicate with one or more intracellular domains of one or more receptors belonging to the superfamily of receptors of the tumor necrosis factor/nerve growth factor tissue (TNF/NGF).

In particular, the present invention relates to a DNA sequence selected from the group including:

(a) cDNA sequence derived from the region encoding the native protein, bind to the intracellular domain of TNF-R;

b) DNA sequences capable of hybridization to DNA (a) under conditions of moderate stringency, and encoding a biologically active protein that binds to the intracellular domain of TNF-R;

(C) DNA sequences which are degenerate with respect to DNA posledovatelem the values, defined in a) and b), as a result of the degeneracy of the genetic code, and which encode biologically active protein, bind to the intracellular domain of TNF-R.

The present invention also relates to a DNA sequence selected from the group including:

(a) cDNA sequence derived from the region encoding the native protein, bind to the intracellular domain of FAS-R;

b) DNA sequences capable of hybridization with cDNA (a) in moderately stringent conditions and which encode biologically active protein, bind to the intracellular domain of the FAS-receptor;

(C) DNA sequences which are degenerate with respect to the DNA sequences defined in (a) and (b), as a result of the degeneracy of the genetic code, and which encode biologically active protein, bind to the intracellular domain of FAS-R.

In specific embodiments, the present invention relates to DNA sequences encoding proteins that are associated with the intracellular domain of the P55 TNF receptor, P75 TNF receptor and FAS-receptor, for example, to DNA sequences coding for the proteins indicated 55.1; 55.3; 55.11; 75.3; 75.16; F2, F9 and DD11.

The present invention also relates to proteins, their analogs or derivatives, coded any of the above paragraph is sledovatelnot of the present invention, and the ability to communicate with one or more intracellular domains of one or more TNF-receptor or FAS-receptor. Variations of this aspect of the present invention include proteins identified 55.1, 55.3, 55.11, 75.3, 75.16, F2, F9 and DD11, their analogues and derivatives thereof.

The present invention also relates to vectors encoding the above proteins of the present invention, containing the above DNA sequence of the present invention; moreover, these vectors can be expressed in the relevant eukaryotically or prokaryotic cells-hosts. In addition, the present invention relates to a transformed eukaryotic or prokaryotic cells-hosts containing these vectors; and to a method of producing proteins, their analogs or derivatives of the present invention by culturing such transformed host cells under conditions suitable for expression of the proteins, with subsequent post-translational modification of these proteins, if necessary, and extraction downregulation of protein, analog or derivative from the culture medium of these transformed cells or from cell extracts of these transformed cells.

In another aspect, the present invention relates to an antibody or to the x active derivatives or fragments, specific to proteins, analogs and derivatives of the proteins of the present invention.

In still another aspect, the present invention relates to the use of DNA sequences of the present invention or the protein of the present invention encoded by those sequences; a use that provides for implementation, in particular, the following methods:

i) modulation method steps TNF or FAS-R ligand on cells carrying a TNF-receptor or FAS-receptor involved in the processing of these cells one or more proteins selected from the group comprising proteins, analogs and derivatives of the present invention, and proteins RS, 55DD FAS-1C or FAS-DD, their analogues or derivatives, where all of these proteins are able to bind with the intracellular domain and modulate the activity of TNF-R or FAS-R, and the said processing cells provides an introduction to these cells of one or more proteins, their analogues or derivatives in a form suitable for intracellular introduction; or the specified processing provides an introduction to these cells a DNA sequence, in the form of a suitable expressing vector, the coding specified specified one or a few proteins, their analogs or derivatives;

ii) mode of action of TNF or FAS-R ligand on cells carrying TNF-R or FAS-R, in which education is Otke these cells antibodies their active derivatives or fragments of the present invention;

(iii) methods of modulating the action of TNF or FAS-R ligand on cells carrying TNF-R or FAS-R, which consists in the treatment of these cells oligonucleotide sequence, the coding sequence which is the antisense, at least part of the sequence of the present invention; or oligonucleotide sequence, the coding sequence which is antisense to the sequence RS, 55DD, FA5-1C or FAS-DD); and the specified oligonucleotide sequence capable of blocking the expression of at least one of the proteins that bind to the intracellular domain of TNF-R or FAS-R;

iv) modulation method steps TNF or FAS-R ligand on cells carrying TNF-R or FAS-R, including:

a) construction of recombinant vector derived from a virus, animals, and carrying a sequence encoding a viral surface protein, which is able to bind to specific receptor on the cell surface, and a sequence selected from the oligonucleotide sequence, the coding sequence which is the antisense, at least part of the sequence of the present invention, and the oligonucleotide sequence, the coding sequence is, which is antisense to the sequence RS, 55DD, FAS-1C or FAS-DD; and when introduced into cells of the indicated virus indicated oligonucleotide sequence capable of blocking the expression of at least one of the proteins that bind to the intracellular domain of TNF-R or FAS-R; and

b) infection of these cells with the vector defined in (a);

v) mode of action of TNF or FAS-R ligand on cells carrying TNF-R or FAS-R, which consists in the treatment of these cells corresponding vector encoding the ribozyme having the sequence specific to a sequence selected from the mRNA sequence that encodes a protein, analog or derivative of the present invention, and the mRNA sequence that encodes RS, 55DD, FAS-1C or FAS-DD); and the specified sequence ribozymes capable of interacting with the mRNA sequence and is able to cleave the specified mRNA sequence resulting in the inhibition expression of the protein, analog or derivative of the present invention or to inhibition of the expression of RS, 55DD, FAS-1C or FAS-DD;

vi) processing method of tumor cells or HIV-infected cells or cells susceptible to other diseases, including:

a) construction of recombinant vector derived from a virus, W is the animals and carrying a sequence encoding a viral surface protein that has the ability to bind to the receptor surface of tumor cells or receptor surface of HIV-infected cells, or which has the ability to communicate with another receptor cell-surface exposed to other violations; and a sequence selected from the sequences of the present invention that encodes a protein, analog or derivative of the present invention, and the sequence coding RS, 55DD, FAS-1C, FAS-DD, or their biologically active analogue or derivative; and the specified protein, analog or derivative of the present invention, or RS, p55DD, FAS-1C, FAS-DD or an analogue or derivative, with the introduction of these tumor or HIV-infected cells or cells with other disabilities, have the ability to destroy these cells;

b) infection of these tumor cells or HIV-infected cells or cells of other violations of the vector defined in (a);

vii) the method of separating and identifying proteins, factors or receptors that can bind with proteins of the present invention, to bind to the intracellular domain, where this method provides for the implementation of affinity chromatography in which the specified protein of the present invention the light is to see with a matrix for affinity chromatography, and this related protein is subjected to contact with a cell extract, then proteins, factors or receptors of this cell extract that are associated with the specified associated protein elute, distinguish and analyze;

viii) the method of separating and identifying proteins that can bind with proteins of the present invention, to bind to the intracellular domain, where the method includes obtaining a yeast twohybrid system, in which the sequence encoding this protein, bind to the intracellular domain, has one hybrid vector and sequence originating from a cDNA library or genomic DNA, is the second hybrid vector, the vectors used to transform yeast host cells, isolated, followed by extraction of the second hybrid vector to obtain a sequence that encodes a protein that is associated with the specified protein, bind to the intracellular domain; and

ix) isolation and identification of a protein able to bind with the intracellular domain of TNF receptor or FAS receptors, providing southern hybridization in mild conditions with subsequent PCR-cloning, in which the sequence of the present invention or part thereof used as a probe for g is britishly with sequences from a cDNA library or genomic DNA library, having at least partial homology with these sequences, and then these hybridized sequence is subjected to amplification and cloning by polymerase chain reaction (PCR), resulting in a gain clones encoding proteins having at least partial homology to these sequences of the present invention.

The present invention also relates to pharmaceutical compositions for the modulation of the action of TNF or FAS-ligand on cells containing as an active ingredient any one component of the following components: (i) the protein of the present invention, or a protein such as RS, 55DD, FAS-1C or FAS-DD, its biologically active fragments, analogs, derivatives or mixture thereof; (ii) a recombinant vector derived from a virus, animal, and encoding a viral surface protein able to bind with the receptors of TNF-R or FAS-R-DD-bearing cells or tumor cells and a sequence encoding a protein, analog or derivative of the present invention, or a sequence encoding RS, p55DD, FAS-1C or FAS-DD; (iii) a recombinant vector derived from a virus, animal, and encoding a viral surface protein defined above in (ii) and oligo-nucleotide sequence encoding the sequence, which is antimyeloma is relative to the sequence RS, 55DD, FAS-1C or FAS-DD; and (iv) a vector encoding the ribozyme sequence capable of interacting with the mRNA sequence that encodes a protein, analog or derivative of the present invention, or to the mRNA sequence that encodes RS, p55DD, FAS-1C or FAS-DD.

In a specific embodiment of the above aspects of the present invention relates to the use of the P55-1C or DNA encoding the P55-1C. This variant of the invention based on the discovery of the fact that the P55-1C could ligand(TNF)-independent manner to induce in the cells of other TNF-associated effects. In accordance with this present invention relates to a method of inducing TNF-associated effects in cells or tissues capable of handling these cells one or more proteins, their analogs or derivatives, where these one or more proteins selected from proteins, all of which are samoassotsiiruyutsya intracellular domains of the p55-TNF-R (P55-1C) or parts of them, are capable of self, and the induction of TNF effect on cells ligand (TNF)-independent manner; and the specified cell treatment involves the introduction into the cells of the indicated one or more proteins, their analogs or derivatives in a form suitable for intracellular introduction or introduction to these cells a DNA sequence, coding is shown one or more proteins, their analogues, or derivatives, in the form of a suitable vector carrying this sequence, and is able to enter this sequence in these cells so that this sequence could be expressed in these cells.

Variants of the above method of the present invention are:

i) method, where the specified cell treatment is carried out by transfection of these cells with the recombinant vector virus-based animal; and the method includes the following stages:

a) constructing a recombinant viral vector carrying a sequence encoding a viral surface protein (ligand)that can bind to specific receptor on the surface of the above treated cells; and a second sequence encoding a protein P55-1C or its fragments, analogs and derivatives, where this protein, its expression in these cells, capable of self-and inducing one or more of TNF-associated effects; and

b) infection of these cells by the vector (a);

ii) method, where the specified TNF-effect induced in these cells is the induction of gene expression of 1L-8, and the specified vector is a vector carrying a sequence encoding, basically, the entire P55-1C, fragments, analogs and the water, who, when their expression in cells capable of self-and signaling to induce the expression of a specified gene 1L-8;

iii) a method of treatment of a tumor or virus-infected cells, or a method of strengthening antibacterial action of granulocytes, where specified viral vector carries a sequence encoding a viral ligand that can bind to specific receptor on the surface of these tumor cells, virus-infected cells or granulocytes, and a sequence encoding specified P55-1C, fragments, analogs and derivatives, which upon expression in these tumor cells, virus-infected cells or granulocytes, induce TNF-associated effects, leading to the death of these cells;

iv) a method of treatment of tumor cells, where specified P55-1C, fragments, analogs or derivatives, with expression in tumor cells, induce the expression of 1L-8, which causes cytolysis of these tumor cells due to their chemotactic activity, attracting granulocytes and other cells to tumor cells and leads, thus, to the death of tumor cells.

In this aspect of the present invention also relates to the intracellular domain of the P55-R (P55-1C) and its fragments, analogs and derivatives used for processing to etok by induction in these cells TNF-associated effects; and his following options:

v) P55-1C, part, analogues and derivatives for use in the treatment of cells by inducing their expression of 1L-8.

vi) the P55-1C, part, analogues and derivatives for use in the treatment of tumor cells by inducing their expression of 1L-8, leading to death of the tumor cells.

In addition, in this aspect the present invention relates to pharmaceutical compositions for the treatment of cells by inducing in them TNF-associated effects, where this composition contains the P55-1C, fragments, analogs, and all their derivatives as the active ingredient, and a pharmaceutically acceptable carrier; and also the following variant of this pharmaceutical composition:

i) a pharmaceutical composition for treatment of cells by inducing in them TNF-associated effects, containing as active ingredient a recombinant vector derived from a virus, animal, and encoding the P55-1C, fragments, analogs, and all their derivatives, and proteins which are capable of contacting a surface protein in the treated cells;

ii) a pharmaceutical composition for treatment of tumor cells, the introduction of which induces the expression of 1L-8, and the subsequent death of tumor cells.

In another aspect of the present izaberete the s refers to soluble oligomeric receptor of the tumor necrosis factor (TNF-R), consisting of at least two samoassotsiiruyutsya hybrid proteins, each of which has (a) at one end of the TNF-binding domain selected from the extracellular domain of TNF-R, its analogs or derivatives; and specified extracellular domain, its analogues or derivatives are not capable of unwanted self and the ability to communicate with TNF; and (b) at the other end, samoassotsiiruyutsya domain selected from (i) mostly complete intracellular domain of the P55-THR (P55-1C), which extends from about amino acid residue 206 to about amino acid residue 426 native molecules of the P55 TNF receptor (P55-R);

(ii) "domain of death" P55-1C extending from about amino acid residue 328 to about amino acid residue 426 native p55-R; (iii) mostly complete intracellular domain of the receptor and FAS/APO1 (FAS-1C); (iv) "domain of death" FAS-1C; and (v) analogs, fragments or derivatives of any of (i)-(iv)with the capacity to self, where specified, at least two samoassotsiiruyutsya protein are self only in the specified ends (b), and at their ends (a) these proteins are able to bind at least two monomers TNF, and each of the ends (a) is able to communicate with one TNF monomer; and, in addition, the present invention relates to salts and functional derivatives specified the CSOs soluble oligomeric TNF-R.

Variations of this aspect of the present invention include all combinations of the above ends (a) ends (b), for example, to those options is soluble oligomeric TNF-R, containing the extracellular domain extracellular domain of the P55-R, and as samoassotsiiruyutsya intracellular domain of the P55 domain-1C.

In addition, the present invention relates to a method for producing a soluble, oligomeric TNF-R, including:

a) constructing expressing vector encoding any one of these hybrid proteins, where the DNA sequence of each of these ends of the hybrid protein derived from cloned DNA sequences encoding mostly complete extracellular domain of the TNF-R, its analogs or derivatives; and from cloned DNA sequences, coding, mostly full P55-1C, "domain of death" P55-1C, FAS-1C, "domain of death" FAS-1C, and their analogs and derivatives; and the resulting ends are ligated to each other, forming the sequence of the hybrid protein, which is then inserted into the specified vector under the control sequences that regulate transcription and translation;

b) introducing a vector in an appropriate host cell, in which is expressed the specified hybrid protein; and

C) purification of the hybrid protein expressed in indicated the cells of the host; moreover, the specified hybrid protein undergoes self before, during, or after the cleaning process, as a result of soluble oligomeric TNF-R.

In addition, the present invention relates to a vector, codereuse the above hybrid proteins used in the above method of the present invention; cell host containing the vector; and to pharmaceutical compositions comprising a soluble, oligomeric TNF-R, its salts or functional derivatives and any mixtures thereof as an active ingredient in combination with pharmaceutically acceptable carrier. Similarly, in accordance with the present invention, soluble oligomeric TNF-R, its salts, functional derivatives and any mixtures thereof can be used to inhibit undesired action of TNF in mammals, and in particular for treating conditions due to excess Fightstar endogenous formed or introduced exogenous TNF; or, alternatively, they can be used for long-term maintenance of a favorable action of TNF in mammalian cells with exogenous introduction of TNF.

In accordance with the above aspect of the present invention should also indicate that it is possible to construct soluble oligomeric receptor FAS/APO1 (FAS-R), which can be COI is used for inhibiting undesirable actions FAS-ligand. Therefore, in another aspect, the present invention relates to soluble oligomeric receptor FAS/APO1 (FAS-R), consisting of at least two samoassotsiiruyutsya hybrid proteins, each of which has (a) at one end, a domain that communicates with FAS-ligand selected from the extracellular domain of FAS-R, its analogues in derivatives, incapable of self and the ability to communicate with FAS-ligand; and b) at another end, samoassotsiiruyutsya domain selected from (i) mostly complete intracellular domain p55-TNF-R (P55-1C), which extends from about amino acid residue 206 to about amino acid residue 426 of native molecules of the P55 TNF-R (P55-R); (ii) "domain of death" P55-1C extending from about amino acid residue 328 to about amino acid residue 426 native P55-R;

(iii) mostly complete intracellular domain of the receptor and FAS/APO1 (FAS-1C); (ivv "domain of death" FAS-1C; and (v) analogues and derivatives of any of (i)-(iv)with the capacity to self, where specified, at least two samoassotsiiruyutsya protein are self only in the specified ends (b), and have the ends (a)which are able to bind at least two monomers FAS-ligand, and each of the ends (a) the ability to communicate with one monomer FAS-ligand; and, in addition, the present invention Rel is referring to the salts and functional derivatives of the specified soluble oligomeric FAS-receptor.

In accordance with this aspect of the present invention also relates to a method for obtaining a soluble, oligomeric FAS-R, including:

a) constructing expressing vector encoding one of dubach these hybrid proteins, where the DNA sequence of each of these ends of the hybrid protein derived from cloned DNA sequences, coding, mostly full extracellular domain of FAS-R, its analogs or derivatives; and from cloned DNA sequences, coding, basically, all the P55-1C, "domain of death" P55-1C, FAS-1c, "domain of death" FAS-1C, all their analogs and derivatives; and the resulting ends are ligated together to form a sequence of hybrid protein, which is then inserted into the specified vector under the control of transcriptional and translational regulatory sequences;

b) introducing a vector in an appropriate host cell, in which is expressed the specified hybrid protein;

C) purification of the hybrid protein expressed in these cells, the owners, and specified a hybrid protein is subjected to self-before, during, or after the cleaning process, as a result of soluble oligomeric FAS-R.

In addition, the present invention relates to an expression vector containing a sequence encoding a soluble the oligomeric FAS-R, used in the above method, the cell host containing a specified vector; and to pharmaceutical compositions comprising a soluble, oligomeric FAS-R, 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 soluble oligomeric FAS-R, its salts or functional derivatives or their mixtures which can be used to inhibit undesirable actions FAS-ligand in mammalian cells, and in particular, for treating conditions due to excess Fightstar endogenous formed or introduced exogenous FAS-ligand.

Similarly to the above description concerning the oligomeric receptors TNP and oligomeric receptors FAS, it is also possible to construct a mixed oligomers, can specifically bind with TNF and FAS-R ligand. Thus, the present invention also relates to mixed oligomeric TNF-R or FAS-R, consisting of at least two samoassotsiiruyutsya hybrid proteins, one of which is selected from any of the above TNF-specific hybrid proteins, and the other is chosen from any of the above FAS-R ligand-specific hybrid proteins, resulting in get mixed oligomer having at least one extracellular domain of the TNF-R, and at least one extracellular domain of FAS-R, United together by self between the intracellular domains or fragments that hybridized with each of these extracellular domains. These mixed oligomeric receptors construct by obtaining, as described above, oligomeric TNF receptors and oligomeric FAS-receptors, and their subsequent mixing, and then selection of the standard ways, those oligomers that are capable of specific binding to FAS-R ligand or TNF. Another way of getting mixed oligomeric receptors is cotransfection suitable host cells with the vectors (described above), encoding any of the TNF-specific hybrid proteins (soluble TNF receptor), and encoding any of the FAS-R ligand-specific hybrid proteins (soluble FAS-receptor); cleanup expressed hybrid proteins that undergo self before, during, and after treatment, as a result of oligomeric receptors; and the following selection with the help of the standard equipment of those oligomeric receptors that have the ability to bind to TNF and FAS-R-the ligands.

Similarly can be obtained pharmaceutical compositions containing mixed oligomeric receptors, their salts or functionality is performance communications derivatives, or any mixture thereof as an active ingredient in combination with pharmaceutically acceptable carrier. In addition, the present invention relates to mixed oligomeric receptors, their salts or functional derivatives or their mixtures which can be used to inhibit undesirable actions of TNF and FAS-R ligand in mammals, and in particular for treating conditions due to excess Fightstar endogenous formed or introduced exogenous TNF and FAS-R ligand; or, alternatively, they can be used for long (extended) maintaining a favorable action of TNF and/or FAS-R ligand in mammalian tissues when exogenous introduction of TNF and/or FAS-R-ligand (soluble form).

Other aspects and variations of the present invention will also be apparent from the following detailed description of the invention.

It should be noted that in the present description, the terms "modulation of the action of TNF on cells and modulation of action of FAS-ligand on cells" mean processing as in vitro and in vivo.

Brief description of drawings

On figa-d schematically depicts a partial and preliminary nucleotide sequence of cDNA clones encoding RS and RS-binding proteins, and deduced amino acid sequence of the protein 55.1, where figure 1(a) shows the sequence (SEQ ID NO: 9) clone 55.11 encoding RS-svyazyvaete protein 55.11; figure 1(b) shows the partial and preliminary sequence (SEQ ID NO: 10 and SEQ ID NO: 11) clone 75.3 encoding RS-binding protein 75.3; and figure 1(C) depicts a partial and preliminary sequence (SEQ ID NO: 12 and SEQ ID NO: 13) clone 75.16 encoding RS-binding protein R (all of these sequences described in Example 1); and figure 1(d) shows the deduced amino acid sequence of the protein 55.11 (SEQ ID NO: 14), obtained on the basis of the nucleotide sequence depicted in figure 1(a)and described in Example 1

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

On figa and 3B presents autoradiogram illustrating the in vitro binding of the protein encoded 55.11 - cDNA with GST-hybrid proteins containing part of the P55-1C; where figa illustrated binding polnorazmernogo protein 55.11 (55.11 - full) with different GST-hybridtype proteins; and figv illustrates the linking part 55.11, coupled with oktapeptidom FLAG, with various GST-hybrid proteins (all of them described in Example 1).

Figure 4 schematically illustrates the comparison of the derived amino acid sequence 55.11 human is a (SEQ ID NO: 14)sequences of related proteins, originating from lower organisms: YHR027c (yeast; SEQ ID NO: 15), SEN3 (yeast; SEQ ID NO: 16), A. thaliana (plant, SEQ ID NO: 17) and .elegans (nematode; SEQ ID NO: 18), as shown in Example 1.

Figure 5 shows a Western blot stained polyclonal anticorodal against MBP, and illustrating the self-Association RS, where the Western blot was obtained from LTO-SDS page gel, which was performed electrophoresis interacting bacterial produced chimeric proteins RS-MBP and RS-GST (lanes 1-4) or control (interaction between the chimeric protein RS-MBP and one GST (lanes 5-8); however, the interaction between the chimeric proteins (and control) was performed on glutathione-agarose beads to perform electrophoresis on SDS page with LTOs, as described in Example 2.

Figure 6 shows phase-contrast micrograph illustrating the cytotoxic effect of a full-sized RS in cells HTta1, expressing transfected with a vector coding for this RS (right panel); and inhibition of this cytotoxic action if the expression vector is blocked by treatment of cells with tetracycline (left panel), as described in Example 2.

Figure 7 illustrates the ligand-independent stimulation Titorenko effect in HeLa cells, transfected with full-P55-R, its intracellular domain, or parts of the intracellular domain, VK is UCA "domain of death", where:

i) in the outermost part 7 schematically shows the different DNA molecules encoding full-P55-R, its intracellular domain fragments of the intracellular domain, which has been integrated into the vector, which was transferrable HeLa cells.

ii) left and middle columns of the graph illustrate the expression of TNF receptor in HeLa cells for each type of receptor, shown in the left part 7, the left column represents the number of receptor in ng/CL sample, and the average column of the graph represents the amount of receptor expressed in units radioiodine TNF associated with transfected cells; and

iii) the right column illustrates the variability of HeLa cells expressing different types of receptors;

and where: all graphs where there's no shading rectangles represent cells transfected into Prilutskii tetracycline, and the shaded rectangles represent cells transfected in the absence of tetracycline (all these options are described in Example 2).

On Fig illustrated ligand-independent induction of gene expression of 1L-8 in HeLa cells, transfected with full-P55-R or its intracellular domain (RS), where on the panel As illustrated by Northern blot analysis RNA extracted from HeLa cells, treated or it is Botanik TNF (two left lanes, marked "control" and "TNF"), and RNA extracted from HeLa cells, transfected with vectors encoding the P55-R, P55-1C or the control protein, luciferase (other tracks marked "P55-1C", "P55-R", and "Luc", respectively); in each case, cells were transfected in Prilutskiy (+) or absence (-) of tetracycline (so for transfected cells, there are two tracks); and where the panel illustrated In staining of 18S rRNA methylene blue in each of the samples of HeLa cells shown in panel A (all of the above options described in Example 2).

Figure 9 (a and b) graphically illustrates the ligand-independent stimulation Titorenko effect in HeLa cells, transfected RV, or its fragments, or FAS-1C, where figa presents the results for p55R, or their fragments, and figv presents the results for FAS-1C. The left panels a and b schematically depicts part 55R or FAS-1C used for transferee, and right on these panels graphically presents the experimental results (all these options are described in Example 2).

Figure 10 schematically depicts a partial and preliminary nucleotide sequence (SEQ ID NO: 19 and SEQ ID NO: 20) cDNA clone (designated F2), which encodes a protein capable of contact RS and FAS-1C, as described in Example 3.

Figure 11 diagram of the automatic depicts a partial and preliminary nucleotide sequence (SEQ ID NO: 21-23) cDNA clone (designated F9) which encodes a protein capable of contact RS and FAS-1C, as described in Example 3.

On Fig schematically depicts a partial and preliminary nucleotide sequence (SEQ ID NO: 24) cDNA clone (designated DD 11), which encodes a protein capable of contact RS, and in particular 55DD and FAS-1C, as described in Example 3.

Detailed description of the invention

In one of its aspects the present invention relates to novel proteins that have the ability to bind to the intracellular domain of the receptors belonging to the superfamily of TNF/NGF, such as TNF receptors (TNF-R) and FAS receptor (FAS-R), and which, therefore, are mediators or modulators of this superfamily of receptors, for example, TNF-R and FAS-R, playing a role, for example, in signal transduction initiated by binding of TNF with TNF receptor and FAS-ligand to the FAS receptor. As examples can serve as proteins that bind to the intracellular domain of the p55-TNF-R (p551C), such as proteins identified in the present description 55.1, 55.3 and 55.11 (Example 1), as well as proteins encoded by cDNA clones F2, R9 and DD11 (Example 3); proteins that are associated with the intracellular domain of the p75-TNF-R, (p751C), such as proteins, designated 75.3 and 75.16 (Example 1); and proteins that are associated with the intracellular domain of FAS-R (FAS-1C), such as proteins encoded by cDNA clones F2, R9 and DD 11 (the example 3). It was found that proteins 55.1 and 55.3 represent parts or fragments of the extracellular domain of the P55 TNF-R (p551C); and other proteins, namely 55.11, 75.3 and 75.16, have not been generally described to the present invention (75.3, 75.16), or they were described (55.11, see, Khan and others, 1992), but their functions or other properties, in particular, on the ability to communicate with TNF-R is not reported (see below, Example 1). New proteins encoded by cDNA clones F2, F9 and DD 11 also represent proteins that have not been previously described, i.e. their sequence are missing in databases with DNA ("GENEBANK") or in databases of amino acid sequences ("PROTEIN BANK").

Thus, the present invention relates to DNA sequences coding for these proteins and to proteins encoded by these DNA sequences.

In addition, the present invention also relates to DNA sequences encoding biologically active analogs and derivatives of these proteins, and analogs and derivatives encoded by those sequences. The receipt of such analogs and derivatives carried out by standard methods (see, for example Sambrook and others, 1989), where the DNA sequences encoding these proteins, can be delegated, added or substituted by one or more codons, so that the resulting analogue had a change in at least one the th amino acid in comparison with the native protein. Acceptable analogs are analogs that retain at least their ability to bind to the intracellular domain of the receptor, which belongs to the superfamily of receptors of the TNF/NGF, such as FAS-R, or TNF-R, for example, RS, RS, or FAS-1C, or which may mediate any other binding or enzymatic activity, such analogs that bind to the P55, RS or FAS-1C, but which do not transmit a signal, i.e., not associated with other downstream receptor, protein or other factor, or not catalyze the signal-dependent reaction. Thus, it can be produced analogs that possess the so-called dominant negative effect, namely analogues that are defective or in respect of the binding, for example, with RS, RS or FAS-1C, or in relation to a subsequent transmission signal after binding. These analogs can be used, for example, for inhibiting the action of TNF or FAS-ligand by competing with natural 1C-binding proteins. Similarly can be produced so-called dominant-positive counterparts, which have the ability to enhance the action of, for example, TNF or FAS-ligand. These analogues have similar or even better 1C-binding properties, as well as similar or even better the ability to transfer with the persecuted, than natural 1C-binding proteins. Similarly derivative can be obtained by standard modifications of the side groups of one or more amino acid residues of proteins or by conjugation of protein with another molecule, e.g. antibody, enzyme, receptor and other molecules, are well known to specialists.

New proteins that bind to the intracellular domain of TNF-R and FAS-R, for example, proteins 55.1, 55.3, 55.11, 75.3, 75.16, as well as proteins encoded cDNA clones P2, F9 and DD11 (also called F2, F9 and DD11), can be used for various purposes, for example:

i) They can be used to simulate or enhance the function of TNF or FAS-R ligand in cases where increased action of TNF or FAS-R is desirable, for example when anticancer, anti-inflammatory or anti-HIV therapy, where you must reach the TNF - or FAS-R ligand-induced cytotoxicity. In these cases, proteins, for example, proteins that bind to RS, such as 5.1, 55.3, and F2, R9 and DD11, and RS (see below and Example 2), and "domain of death" RS (55DD), which enhances the action of TNF; or proteins F2, R9 and DD11, and FAS-1C and FAS-DD, to enhance the effect of FAS-R ligand, i.e. possess cytotoxic activity, can be introduced into the cell using standard techniques known per se. For example, since e and proteins are intracellular and because it is desirable, so they were put only in those cells where it is preferable to induce the action of TNF or FAS-R ligand, for specific introduction of these proteins in cells, you must use a specific system. One way of constructing such a system is the creation of a recombinant animal virus, for example, virus, derived from cowpox virus, to obtain DNA from which can be extracted and sequentially introduced two genes: the gene encoding a ligand that binds to cell surface proteins expressed by the cells, such as protein Dr virus SPEED and (HIV), which specifically binds to certain cells (CD4-lymphocytes and related cells leukemia), or the gene encoding another ligand that specifically binds to cells bearing TNF-R or FAS-R, so that the recombinant viral vector was capable of binding to cells bearing TNF-R or FAS-R; and the gene encoding a new protein, bind to the intracellular domain, or a protein RS, 55DD, FAS-1C or FAS-DD. Thus, a protein on the surface of the virus binding to the cell surface, will be specific to transmit the virus to a tumor cell or other cell carrier TNF-R or FAS-R, after which the sequence encoding the protein to bind to the intracellular domain, or in series is here, coding RS, 55DD, FA8-1C or FAS-DD, will be introduced into the cells with this virus, and after its expression in these cells is the induction enhanced activity of TNF or FAS-R ligand, leading to the death of tumor cells, or other cells bearing TNF-R or FAS-R, the destruction of which would be desirable. The design of such a recombinant animal virus can be carried out using standard techniques (see, for example, Sambrook and others, 1989). Another possibility of the introduction of these proteins in cells can be realized by introducing sequences of these new proteins or RS, p55DD, FAS-1C or FAS-DD in the form of oligonucleotides that can be absorbed by cells and expressed in them.

ii) They can be used for inhibiting the action of TNF or FAS-R ligand, for example, in cases of tissue destruction in the septic shock, rejection reactions of the type "graft versus host", or acute hepatitis, where you want to block TNF-induced intracellular signaling TNF-R or FAS-R ligand-induced intracellular signaling FAS-R. In this case, for example, to enter the cells, using standard techniques, the oligonucleotides having antisense coding sequences for these new proteins or antisense coding sequence for RS, P55DD, PAS-1 or FAS-DD, that would effectively block the translation of mRNA encoding these proteins, and thus block their expression, which, ultimately, would lead to the inhibition of TNF or FAS-R ligand.

Such oligonucleotides can be introduced into cells using the above virus; and the second sequence contained in the virus, should be oligo-nucleotide sequence. Another way is the use of antibodies against these proteins, leading to inhibition of their ability to intracellular signal transmission. It is possible that these new proteins are extracellular domain and intracellular domain, and the intracellular domain binds to TNF-R or FAS-R-binding domain, and then antibodies generated against the extracellular domain, can be used to block TNF or FAS-R-associated functions.

Another way of inhibiting the action of TNF or FAS-S-ligand is a recently developed method using ribozymes. Ribozymes are catalytic RNA molecules that specifically cleaved RNA (i.e. have the property of autocatalysis). Ribozymes can be designed to break down the desired RNA, for example mRNA, encoding a new protein of the present invention, or mRNA encoding RS, 55DD, FAS-1C or FAS-DD. Such is isozyme must have the sequence - specific mRNA, and must be able to interact with it (complementary binding) with subsequent cleavage of this mRNA, which should ultimately lead to a reduction (or complete inhibition) in the expression of the protein, which is necessary to inhibit, and the reduction of the expression depends on the level of expression of ribozymes in the target cell. For the introduction of ribozymes in cells (e.g., in cells carrying TNF-R or FAS-R) can be any suitable vector, e.g. a plasmid, viral animal vectors (retroviruses, and vectors that are commonly used for these purposes (see above, where the virus has, as a second sequence of cDNA encoding a selected sequence of the ribozyme).

In addition, can be designed ribozymes with multiple targets (multi-ribozymes), which may be used, for example, for inhibiting expression of one or more proteins of the present invention and/or RS, 55DD, FAS-1C or FAS-DD (review method, etc. dedicated to ribozymes, see Chen et al., 1992; Zhao & Pick, 1993; Shore et al., 1993; Joseph & Burke, 1993; Shimayama et al., 1993; Cantor et al., 1993; Barinaga, 1993; Crisell et al., 1993; Koizumi et al., 1993).

iii) They can be used for isolation, identification and cloning of other proteins able to contact them, such as other proteins, participated is participating in the process of signal transmission, below from the intracellular domain of TNF-R or FAS-R. In this case, these options, namely DNA sequences encoding these proteins, can be used in yeast twohybrid system (see Example 1), in which the sequence of these proteins can be used as "bait" for the selection, cloning and identification of cDNA libraries or genomic DNA of other sequences ("production"), encoding proteins that might be associated with these new proteins, bind to the intracellular domain of TNF-R or FAS-R in a Similar way can also be determined whether specific proteins of the present invention, namely the proteins that are associated with RS, RS or FAS-1C, contact with other receptors of the superfamily of receptors of the TNF/NGF. For example, it was recently reported (Schwalb and others, 1993; Baens to others, 1993, Crowe and others, 1994)that in addition to the P55 and p75-TNF-R, there are other TNF receptors. In line with this, using yeast twohybrid system, it is possible to accurately check whether the proteins of the present invention the ability of specific binding to these other TNF receptors or other receptors of the superfamily of TNF/NGF. In addition, this method can also be used to determine whether proteins of the present invention the ability to communicate with other known re what atrami, in the activity which they can play a functional role.

iv) These new proteins can also be used for isolation, identification and cloning of other proteins of the same class, i.e. proteins, bind to the intracellular domain of TNF-R or FAS-R or functionally related receptors, and are involved in intracellular signal transmission. In this case, it may be used the above-described yeast dvuhserijnaya system, or can be used a recently developed (Wilks and others, 1989) system using fuzzy southern hybridization with subsequent PCR cloning. In the publication Wilks and others describes the identification and cloning of two suspected protein-tyrosine-kinase using a soft southern hybridization with subsequent cloning by PCR, based on the known sequence of motif kinase alleged kinase sequence. In accordance with the present invention, this method can be applied using sequences of new proteins for the identification and cloning of proteins related proteins, bind to the intracellular domain of TNF-R, FAS-R, or a related receptor (receptor superfamily, TNF/NGF).

v) In yet another variant, the new proteins of the present invention can be used in the methods affine chromatog is the her for isolation and identification of other proteins or factors, with whom they can contact, such as other receptors, related to TNF-R (receptor superfamily, TNF/NGF) or other proteins or factors involved in the process of signal transmission. In this case, the proteins of the present invention may be separately associated with the matrix used for affinity chromatography and then subjected to contact with cell extracts or selected proteins or factors that are suspected of involvement in the transmission of intracellular signals. After carrying out affinity chromatography other proteins or factors that are associated with the new proteins of the present invention, can be polyuretane, isolated and characterized.

vi) As mentioned above, the new proteins of the present invention can also be used as immunogens (antigens) to produce antibodies against them. These antibodies can also be used for cleaning of new proteins either from cell extracts or from producing their transformed cell lines. In addition, these antibodies can also be used for diagnostic purposes of identifying disorders related to abnormal functioning of the system TNF or FAS-R ligand, for example, the cellular effects induced by overactive or inactive TNF or FAS-R-ligands. For example, in the case where the hat a disorder associated with insufficient functioning of the transmission system of the intracellular signal, involving new proteins, these antibodies may serve as an important diagnostic tool.

It should be noted that the isolation, identification and characterization of new proteins of the present invention can be implemented using well-known standard methods of screening. For example, one such method of screening (method of obtaining 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 can be used other well-known methods, such as affinity chromatography, DNA hybridization, and the like for separation, identification and characterization of new proteins of the present invention, or for separation, identification and characterization of other proteins, factors, receptors, etc. with the ability to communicate with the new proteins of the present invention or with receptors belonging to the family of receptors of the TNF/NGF.

As for the above-mentioned antibodies, the term "antibody"used in the present description, refers to polyclonal antibodies, monoclonal antibodies (mAb), chimeric antibodies, antiidiotypic antibodies (anti - 1d), against antibodies that can be labeled in soluble or bound form, as well as their fragments obtained used the eating techniques such as enzymatic cleavage, peptide synthesis or technique of recombinant DNA, etc.

Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with the antigen. Monoclonal antibodies represent a largely homogeneous population of antigen-specific antibodies, which are basically similar epilepsyusa sites. Monoclonal antibodies can be obtained by known methods. See, for example, Kohier & Milstein, Nature, 256: 495-497 (1975); U.S. patent No. 4376110; Ausubel et.al., eds., Harlow &Lane, ANTIBODIES, A LABORATORY MANUAL, Gold Spring Harbor Laboratory (1988); u Colligan et al., eds., Current Protocols in Immunology, Creene publishing Assoc. & Wiley Interscience, N. Y., (1992, 1993), the contents of these works is introduced in the present description by reference. These antibodies can be of any immunoglobulin class 1qG, DM, 1q, 1gA, gild, and any of their subclasses. Hybridoma producing monoclonal antibodies of the present invention can be cultured in vitro, in situ or in vivo. The possibility of producing high titers of monoclonal antibodies in vivo or in situ makes this method particularly preferred.

Chimeric antibodies are molecules composed of different parts originating from different animal species, for example, such molecules, which have a variable region derived from mouse on the different mAb, and a constant region derived from human immunoglobulin. Chimeric antibodies are used mainly to reduce immunogenicity and increase output for producing such chimeric human/murine mAb can be used in cases when the mouse mAb have increased the outputs of the hybrid, but have increased immunogenicity to humans. Chimeric antibodies and methods for their preparation are known and described in the literature (Cabilly et al., Proc.Natl.Acad. Sci.USA 81: 3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984); Boulianne et al., Nature 312: 643-646 (1984); Cabilly et al., application for European patent application 125023 (published November 14, 1984); Neuberger et al., Nature 314: 268-270 (1985); Morrison et al. Application for Europatent 173494 (publ. on March 5, 1986) Taniguichi et al., application for European patent application 173494 (published March 5, 1986), Neuberger et al., PCT application WO 8601533 (published March 13, 1986); Kudo and other application for the European patent application 184187 (published June 11, 1986); Sahagan et al., J. Immunol. 137: 1066-1074 (1986); Robinson and others, the application for international patent WO 8702671 (published may 7, 1987); Liu et al., Proc. Natl.Acad. Sci. USA 84: 3439-3443 (1987); Sun et al., Proc. Natl.Acad. Sci. USA 84: 214-218 (1987); Better et al., Science 240: 1041-1043 (1988); and Harlow &Lane, ANTIBO DIES: A LABORATORY MANUAL (see above). All these works are introduced in the present description by reference.

Antiidiotypic (anti-1d) antibody is an antibody that recognizes antigenic determine what you mainly associated with antigennegative centre antibodies. Idiotypical antibody can be obtained by immunization of an animal of the same species and genetic type (e.g., a mouse line), and the source of the mAb, monoclonal antibody, which produce anti-1d antibody. Immunized animals can recognize and respond to idiotypical determinants of the immunizing antibody by producing antibodies against these idiotypical determinants (anti-1d antibody). See, for example, U.S. patent 4699880, which is introduced into the present description by reference.

Antiidiotypic antibody can also be used as an immunogen to induce an immune response in another animal, producing a so-called anti-anti-idiotypic antibody. This anti-anti-idiotypic antibody may be on its antigenic determinants are identical to the original mAb that induces antiidiotypic antibody. Thus, by using antibodies to idiotypical determinants mAb can be identified other clones expressing antibodies of identical specificity.

In accordance with these monoclonal antibodies produced against 1C-binding proteins, their analogs or derivatives of the present invention or against RS, 55DD, FAS-DD 1C, FAS-DD, and is Logov or derivatives can be used to generate antiidiotypic antibodies in appropriate animals such as mouse BALB/c mice. The spleen cells from these immunized mice are used for the production of anti-1d hybridomas secreting anti-1d mAb. In addition, these anti-1d mAb can be conjugated with a carrier, such as hemocyanin lymph snails (KLH)and used to immunize other BALB/c mice. Sera from these mice will contain anti-anti-idiotypic antibodies having binding properties of the original mAb specific for the epitope of the above 1C-binding protein, its analogs or derivatives, as well as RS, 55DD, FAS-1C or FAS-DD, their analogs or derivatives.

Thus, anti-1d mAb have their own idiotypical epitopes or "idiotype"structurally similar to the estimated epitope, such as protein-α GPB.

The term "antibody"used in the present description, means also intact molecules and fragments thereof, such as Fab and F(ab')2that are able to bind to the antigen. Fab and F(ab')2the fragments are part of intact antibodies without Fc-fragment, and more rapidly eliminated from the bloodstream and have a smaller tkanespetsificheskie binding than an intact antibody (Wahl et al., J. Nucl. Med. 24: 316-325 (1983).

It should be noted that Fab, F(ab')2and other fragments used in the present invention, can be used for detection and quantitative assessment of the 1C-binding proteins or RS, 55DD, FAS-1C or FAS - DD in accordance with the methods described in this application for intact antibody molecules. Typically, these fragments produced by proteolytic cleavage with the use of enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2-fragments).

It is believed that the antibody is capable of contact with the molecule in the case, if it is able to engage in specific response to this molecule, in which this molecule binds to the antibody. The term "epitope" means that portion of any molecule capable of contacting the antibody, which can be recognized by this antibody. Epitopes or antigenic determinants usually consist of chemically active surface groups of molecules, such as groups of amino acid residues or polysaccharide side chains and have specific three-dimensional structure, as well as specific charge.

"Antigen" is a molecule or part of it, are able to bind with the antibody and is able, moreover, to induce the animal the production of antibodies which are capable of contacting the epitope of this antigen. The antigen may have one or more epitopes. The term "specific reaction antigen" is meant that the antigen can react at high selectivity compliance with the criterion him antibody and does not react with many other antibodies, produced by other antigens.

Antibodies, including antibody fragments used in the present invention, can be used for quantitative or qualitative detection 1C-binding proteins or RS, p55DD, FAS-1C, FAS-DD in the sample, or to detect prihodti cells that Express 1C-binding proteins of the present invention or proteins RS, 55DD, FAS-1C and FAS-DD. This detection can be carried out using immunofluorescent techniques using a fluorescently labeled antibody (see below) in combination with the methods of optical microscopy, flow cytometry, or fluorometry.

The antibodies (or fragments thereof)used in the present invention, can be used histologically in immunofluorescence or immunoelectron microscopy, for in situ detection 1C-binding proteins of the present invention or RS, 55DD, FAS-1C, FAS-DD. In situ - Detection can be done by taking a tissue sample from the patient and the introduction of a labeled antibody of the present invention in a sample. The antibody (or fragment) preferably by spraying or coating this labeled antibody (or fragment) of the biological sample. Using this method, you can determine not only fightstyle 1C-binding proteins or RS, 55DD, FAS-1C, FAS-DD, but also to assess the races is the definition of these proteins in the tissue studied. Any specialist it is obvious that with the use of the present invention can be modified in any histological methods a wide range (for example, methods of staining) for the implementation of such in situ detection.

These analyses fightstyle 1C-binding proteins of the present invention or RS, p55DD, FAS-1C, FAS-DD usually carried out by incubating a biological sample, such as a physiological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in Prilutskii detected labeled antibody capable of identifying 1C-binding proteins or RS, 55DD, FAS - 1C, FAS-DD, and the subsequent detection of this antibody by any of the existing standard methods.

The biological sample may be immobilized on a solid medium such as microcellulose or other solid-phase carrier, capable immobilizative cells, particles, cells or soluble proteins. This media can then be washed with appropriate buffer, and then processed detektivami labeled antibody in accordance with the present invention, as described above. Solid-phase carrier may be again washed with buffer to remove unbound antibodies. After that, the amount of bound label n is specified solid carrier or substrate can be determined by standard methods.

The term "solid substrate", "solid-phase media", "solid support", "solid substrate", "substrate" or "carrier" refers to any substrate or any medium that is capable of contact with the antigen or antibody. Well-known substrates or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified cellulose, polyacrylamides, gabbros, and magnetite. By its nature, this carrier may be soluble to some extent or insoluble depending on the purpose of the present invention. The material for the carrier can have virtually any configuration, provided that the immobilized thereon a molecule capable of contacting an antigen or antibody. For example, the substrate or the carrier may have a spherical balls) and cylindrical (in the form of the inner surface of the test tube) configuration, or to constitute the outer surface of the rod. Alternatively, the surface of the carrier may be flat such as a sheet, strip, etc. Preferred carriers are polystyrene beads. However, it should be noted that by routine experimentation, any specialist can find suitable carriers for binding of the antibody or antigen.

Linking active is th this antibody, as described above, may be determined by known methods. Each specialist through routine experimentation can determine working and optimal conditions of analysis for each case.

In each case the analysis can be further implemented other stages, such as washing, stirring, filtering, etc. if necessary.

One of the ways of getting detected labeled antibody of the present invention involves the binding of this antibody to the enzyme followed by the enzyme-linked immunosorbent assay (ELISA). This enzyme, in turn, can then be subjected to the interaction with the corresponding substrate, so that the result of this reaction, the substrate was producirovanie chemical molecule or group, which could be detected by spectrophotometry, fluorometry or by visual observation. Enzymes that can be used to obtain a program labeled antibodies, are (but are not limited to) malatdegidrogenaza, staphylococcal nuclease, steroid-S-isomerase, yeast alcoholdehydrogenase, alpha-glycerol dehydrogenase, triazolopyrimidine, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. The detection antibody can be performed colorimetric methods using a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of the substrate with the standards, made in the same way.

Detection can also be implemented using any other immunoassay. So, for example, radioactively labeled antibodies or fragments thereof can be detected by R-Rtrate using radioimmunological assay (RIA). A good description of the RIA provides Work T. and others (Laboratory Technigues and Biochemistry in Molecular Biology; Noeth Holland Publishing Company, NY (1978) with reference to the Chapter entitled " An Introduction to Radioimmune Assay and Related Technigues", Chard, Etc; this work is introduced in the present description by reference. The radioactive isotope can be detected using a gamma counter, scintillation counter, or by using autoradiography.

In accordance with the present invention the antibody can also be labeled with a fluorescent compound. If a fluorescently labeled antibody subjected to light irradiation with the appropriate wavelength, Fightstar this antibody can be detected due to fluorescence. The most common is a connection, used for fluorescent labeling, are isothiocyanate fluorescein, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine.

The antibody for its detection, can also be marked using fluorescent metals, such as E or other metals of the series of lanthanides. These metals can be linked to the antibody through groups, forming chelate complexes of metals, for example, such groups as diethylenetriaminepentaacetic acid (ETP).

For their detection antibody can also be labeled by binding chemoluminescent connection. Fightstyle such chemiluminescence-labeled antibodies can then be detected by the presence of luminescence produced by the chemical reaction. Examples of suitable chemiluminescent compounds tags are luminal, isoluminol, heat-resistant ester acridine, imidazole, salt acridine and an ester of acridine and oxalic acid.

Similarly, a bioluminescent compound can also be used to tag the antibodies of the present invention. Bioluminescence is a type of chemiluminescence observed in biological systems, where the catalytic protein increases the efficiency of the chemiluminescent reaction. Fightstyle bioluminesce the spas protein can be detected by the presence of luminescence. Suitable for marking bioluminescent compounds include luciferin, luciferase and akwari.

The antibody molecule of the present invention may be adapted for use in immunometric analysis, known as the "two-layer" (or "two-step"analysis or "sandwich"assay. In a typical immunometric the analysis of a certain number of its antibody (or fragment) associated with the solid substrate or solid carrier and add a certain number of detected labeled soluble antibody, and then analyzed by Fightstar and/or determine the amount of ternary complex formed between the antibody immobilized on a solid phase carrier, antigen, and labeled antibody.

Typically and preferably immunodeficiency analysis is a "direct" analysis, in which the antibody associated with the solid phase carrier first subjected to contact with the test sample for extraction of antigen from the sample by formation of a double complex antibody on the solid phase carrier - antigen". After incubation for an appropriate period of time solid support is washed to remove residual liquid sample, including unreacted antigen, if any, and subjected to contact with a solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule"). After the second incubation period, during which the labeled antibody forms a complex with the antigen bound to the solid phase through its antibody, the solid support is washed a second time in order to remove the unreacted labeled antibody.

In the "sandwich"-the analysis of another type, which can also be applied to the antigens of the present invention, use of the so-called "simultaneous" and "reverse" assays. Simultaneous analysis provides one stage of incubation, because the antibody associated with the solid carrier and the labeled antibody is added to the test sample at the same time. After completion of incubation, the solid support is washed to remove residual liquid sample and the labeled unconjugated antibodies. Then fightstyle labeled antibody associated with the solid carrier, define as well as in the "direct" analysis described above.

In "reverse" analysis first, add a solution of labeled antibody to the liquid sample, and then after a suitable period of incubation, add its antibody associated with the solid carrier. After the second incubation the solid phase was washed in a standard way to remove residues of the test specimen and solution of unreacted labeled antibody. The definition of labeled antibody, is knitted with a solid carrier, carried out as in "simultaneous" and "direct" tests.

New proteins of the present invention, after their selection, identification and characterization of any of the standard methods of screening, for example, a method using yeast double-hybrid, affinity chromatography and other known techniques can then be produced by standard methods of recombinant DNA (see, for example, Sambrook and others, 1989), in which a suitable eukaryotic or prokaryotic cells-owners transform appropriate eukaryotic or prokaryotic vectors containing sequences encoding proteins. In accordance with this present invention also relates to the specified expressing vectors and transformed cells-owners. As mentioned above, these proteins also include their biologically active analogs and derivatives, and therefore the present invention also relates to vectors coding for the analogues of these proteins; and to transformed cells-owners that produce these analogues. Derivatives of the proteins produced by standard modifications of proteins or their analogues produced by the transformed cells of the host.

In another aspect the present invention relates to the use of separate intracellular domain of the p55-TNF-R (RS) or FAS-R (FAS-1C) Ilyich so-called "domain of death" (p55DD) or FAS-DD), accordingly, as agents for intensifying action of TNF or FAS-R ligand on cells, independently from each other (see Example 2). If these cells, for example cancer or HIV-infected cells, it is necessary to generate TNF - or FAS-R ligand-induced cytotoxic effect in these cells can be introduced RS, 55DD or FAS-DD using the above methods (see above p.(i)) using recombinant animal virus (e.g. vaccinia virus). In addition, in the present invention can be used native RS, 55DD, FAS-1C or FAS-DD, their biologically active analogs, derivatives or fragments, all of which can be obtained by the methods described above.

Similarly, the present invention also relates to the specific blocking of TNF-or FAS-R ligand action by inhibiting the activity RS, 55DD, PAS-1C or FAS-DD, for example, by introducing into cells of antisense oligonucleotide, resulting in blocking the expression of RS, 55DD, PAS-1C or FAS-DD.

The present invention also relates to pharmaceutical compositions containing recombinant vectors based on animal viruses encode proteins that bind to the intracellular domain of TNF receptor or FAS (including RS, 55DD, FAS-1C and FAS-DD), and encoding a viral surface proteins capable of specific the key contact with the surface protein of the target cells (for example, cancer cells), resulting in these vectors provide a directional insertion sequences of proteins that bind to the intracellular domain, in the right cell.

In another aspect, the present invention relates, in particular, to the effects of self-intracellular domain of the P55 TNF receptor (P55-1C, see Example 2). An example of such effects, which are usually mediated by the binding of TNF with its receptor and simulated signalpeptide activity samoassotsiiruyutsya P55-1C or parts thereof, is inducing the expression of the gene encoding 1L-8.

Interleukin-8 (1L-8) is a cytokine that belongs to the subclass of chemokines, which has expressed chemotactic activity, and, as we have seen, plays an important role in the chemotaxis of granulocytes and cells of other types associated with several pathological conditions (see, for example, Endo and others, 1994; Sekido and others, 1993; Harada and others, 1993; and Ferrick and others, 1991).

The tumor necrosis factor (TNF) has a useful activity, and therefore it can be used directly for the destruction of tumor cells and virus-infected cells or to enhance the antibacterial activity of granulocytes. However, as mentioned above, TNF also has undesirable activity, and in these cases its activity, it is desirable to block, including t the cases when using high doses of TNF, such as when anticancer, antiviral and antibacterial therapy.

Accordingly, it is desirable to develop a method that would allow to regulate the activity of TNF or to obtain such a connection, which would be able to simulate the useful activity of TNF in cells or tissues requiring special handling.

In accordance with the present invention it was found that samoassotsiiruyutsya intracellular domain of the P55-R (P55-1C) may, ligand-independent way, we can simulate some of the effects of TSR, for example, "domain of death" P55-1C can induce cytotoxic effects in cells, and the P55-1C can induce gene expression 1L-8. Thus, using the P55-1C, it is possible to simulate the function of TNF by site-directed mechanism, i.e. you can enter the P55-1C only in those cells or tissues that need processing.

One example of the aforementioned methods can serve as specific transfection or transformation) of tumor cells or malignant tissue DNA molecule encoding the P55-1C or its fragment, which can not only induce cytotoxic effects on these cells or tissues, but also to enhance these effects by co-induction 1L-8, which will result in the accumulation of these cells or tissue granulocytes or other lymphocyte is in, which, in turn, can contribute to the destruction of tumor cells or tissue. This method avoids the need for the introduction of high doses of TNF associated with undesirable side effects.

Using standard techniques of recombinant DNA can be obtained from different parts of the P55-1C and determined which region is responsible for each TNF-induced effect. For example, it was found that the "domain of death" responsible for cytotoxicity (Example 2), and were obtained from various other designs, containing part of the P55-1C, which (together with part or full "domain of death") may be responsible for other TNF-effects and which can be used, a ligand-independent manner, after their self, in order to induce these effects, for example, induce the expression of 1L-8.

It should be noted that the sequence of the P55-1C is involved in the induction of other TNF-associated effects (e.g. induction 1D-8), may differ from the sequence, which is responsible for cytotoxicity, i.e. it may not have, or only have part of the "domain of death", and to have other motives originating from other areas of the intracellular domain, or it can itself be a sequence of these other distinctive attributes sequence (sequence what elnett other "motive"), involved in the induction of other effects.

Accordingly, as described above and below can be obtained by expressing the vectors containing the specified region of the P55-1C, their analogues or derivatives, and expressed in the cells of the host, and then purified and analyzed for their activity. This method can be obtained a number of fragments of the P55-1C, having one or more TNF-associated effects, and these fragments can be used in various ways for the treatment of pathological conditions of any number, for example, viral infections, bacterial infections, tumors, etc. In all these cases, the specific activity of these fragments can be strengthened by joint injection (or co-transfection) fragment P55-1C responsible for the induction of gene expression of 1L-8 that will produce the desired chemotactic activity 1L-8 and increased destruction of cells or tissues that need to destroy.

Thus, without resorting to system introduction TNF can induce its beneficial effects by the specific introduction of all the P55-1C or its fragment in cells or tissues that are intended for processing.

the P55-1C can be specifically introduced into the cells or tissue for their destruction using any of the above ways. For example, one of these ways is s provides for constructing a recombinant animal virus (for example, derived from the cowpox virus)DNA which enter the following two gene: a gene that encodes a ligand that binds to cell surface proteins specifically expressed by cells, such as protein Dr virus SPEED and that specifically binds to certain cells (CD4 lymphocytes and related cells leukemia); or any other ligand that specifically binds to cells bearing TNF-R, and through which a recombinant viral vector can contact these TNF-R-bearing cells; and the gene encoding the P55-1C or its fragment. Thus, expression of the protein, to bind to the cell surface, on the surface of the virus is specific to transmit the virus to a tumor cell or other TNF-R - carrier cell, resulting sequence encoding the P55-1C or its fragment, will be using this virus, introduced into cells and subsequent expression of this sequence in these cells will lead to increased TNF-effect, contributing to the destruction of tumor or other TNF-R-bearing cells, which must be destroyed, or inducers, such as the expression of 1L-8, leading to the deaths of these cells. The design specified recombinant animal virus can be carried out using standard techniques (see, for example, Sambrook and others, 1989). The other is the second method involves the introduction of sequences P55-1C or their fragments in the form of oligonucleotides, which can be absorbed by cells and expressed in them.

The present invention also relates in particular to pharmaceutical compositions containing the above recombinant viral vectors encoding the P55-1C or its fragments, as well as encoding the viral surface protein that can specifically bind to surface proteins in the target cells (e.g. cancer cells), resulting in the specified vector provides the insertion sequence P55-1C or its fragments in these cells.

In another aspect the present invention relates to new synthetic TNF-receptors, which are soluble and are capable of oligomerization with the formation of dimeric, and possibly higher order multimeric TNF-receptor molecules, each monomer of which is capable of contacting the monomer TNF. Native TNF is homotrimer containing three active TNF-monomer, each of which is able to communicate with one molecule of the receptor; whereas the natural receptor TNF represent owl monomers, each of which is able to communicate with only one monomer of homotrimeric TNF molecules. Thus, if TNF binds to TNF receptors on the cell surface, he is able to communicate with three receptor molecules, th is leads to the formation of TNF-receptor clusters, which, obviously, initiate the process of signal transmission, which leads, ultimately, to the induction of the observed TNF-effects.

Although TNF has many useful effects, such as the ability to the destruction of unwanted cells, such as, tumor cells or virus-infected cells, as well as increased antibacterial activity of granulocytes, however, TNF has many undesirable effects, such as causing severe diseases, including autoimmune disorders, rheumatoid arthritis, rejection reaction type "graft versus host (graft rejection) and septic shock; and it is suspected that TNF is the main cause of the pathological destruction of tissue. TNF can also cause excessive loss of body mass (cachexia) by suppressing the activity of adipocytes. In addition, even with the introduction of TNF in order to use its useful activity, for example, in the treatment of various malignant tumors, viral diseases, the used doses of TNF are often quite high and cause the patient a number of undesirable cytotoxic side effects, such as the destruction of healthy tissue.

Therefore, in all the above cases, where the action of TNF is unfavorable, it is desirable to use an effective inhibitor of NF. We developed many of TNF-blocking agents, including soluble proteins, which possess the ability to bind to TNF and inhibiting the binding of TNF to its receptors and thereby inhibiting the cytotoxic action of TNF (see EP 308378, EP 398327 and EP 568925). However, these TNF-binding proteins or soluble TNF-receptors are Monomeric, and therefore each of them is able to communicate only with one of the monomers of homotrimer TNF. Therefore, blocking the function of TNF in these proteins may not be complete, since each molecule TNF-related Monomeric receptor, has two free TNF-monomer that is able to communicate with TNF receptors on the cell surface and thereby contribute to the adverse effects of TNF on cells.

Therefore, to solve the problems associated with blocking the function of TNF in accordance with the present invention a method was developed that allows to construct hybrid proteins, namely soluble oligomeric TNF-receptors, which have the ability to contact at least two monomers native homotrimeric molecules TNF. In accordance with this method, these soluble oligomeric TNF receptors are associated with their TNF ligand with higher avidity than known previously obtained soluble TNF - binding proteins or receptors. For example, if RA is soluble TNF-receptor of the present invention was obtained in the form of a dimer, he is able to contact the two monomers of the TNF trimer, resulting in a more complete and longer lasting neutralization of TNF due to a lower rate of dissociation of the dimeric soluble receptors of TNF. 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 big advantage because of the likelihood that their excretion from the body will be slower.

A prerequisite for the development of soluble oligomeric TNF receptors of the present invention was the discovery of the fact that the intracellular domain of the P55 TNF-receptor has the ability to samoassotsiatsiyu, and the fact that this intracellular domain (P55-1C) is part of the so-called "death domain", which also has the ability to self-and which directly, ligand-independent manner, may cause cytotoxic effects on the cells (see Example 2). Using this property to self-intracellular domain (P55-1C) and its "domain of death", it is possible, using standard techniques of recombinant DNA to construct a hybrid protein containing, basically, the entire extracellular domain of the TNF receptor, such as receptor P75-R or P55-R, and preferably the P55-R, and sewn with him, mainly ve the R intracellular domain (P55-1C) or "domain of death". Thus, it is possible to construct a new hybrid product, which at its one end has a TNF-binding domain, i.e. the extracellular domain of the receptor, and at its other end has an intracellular domain, or "domain of death", are capable of self. Accordingly, this product will have the ability to oligomerization through self between his two (and possibly more) intracellular domains, or "domain of death"that will lead to the formation of oligomers (or at least dimers)that contains at least two domains, bind to the TNF.

In addition, in accordance with the present invention it was also found that the receptor FAS/APO1 has samoassotsiiruyutsya intracellular domain, including samoassotsiiruyutsya "domain of death", with a certain homology with the P55-1C and its "domain of death" (Example 2). Therefore, in accordance with the present invention can be constructed of soluble oligomeric TNF-receptors by hybridization of the extracellular domain of TNF receptor (described above) with the intracellular domain, or "domain of death" FAS/APO1 receptor.

In both the above cases, oligomeric TNF-receptors of the present invention are soluble due only to the fact that they are soluble extracellular domain of the receptor TYR and soluble Nutrilite the hydrated domain or its "domain of death" or receptor p55-TNF, either receptor FAS/APO1, i.e. they do not contain transmembrane (insoluble) domain of any of these receptors.

Detailed description of the construction of the above oligomeric TNF receptors of the present invention are provided below in the Example. However, it should be noted that after the construction of the oligomeric TNF receptors of the present invention, a situation may arise that were not previously described, in which the extracellular domain of the TNF receptor will be able to self, and this situation may be undesirable, since it will inhibit the ability of oligomeric receptor to contact two or more monomers homotrimeric molecules TNF, or it can lead to less than optimal binding of these monomers TNF. Therefore, to avoid such situations by using standard techniques of recombinant DNA to modify the extracellular domain of the TNF receptor, e.g., by deletion or substitution of one or more amino acid residues contained in samoassotsiiruyutsya region, in order to prevent possible self. This modification of the extracellular domain of the TNF-receptor is therefore also part of the present invention, and modified so the domains are analogues or derivatives of the extracellular domain of the TNF receptor. Similar treatment is ω samoassotsiiruyutsya intracellular domain (1C) or "death domain" (DD) receptor P55-DD or receptor FAS/APO1, used in the oligomeric TNF receptors of the present invention can also be analog or derivative of the natural domain, i.e. it can be any modification of the sequence P55-1C or its fragments, including "death domain" (P55-DD) or any modification of the sequence of the intracellular domain of FAS-1C) or its fragments receptor FAS/APO1, including "death domain" (FASDD), provided that as a result of these modifications is formed samoassotsiiruyutsya product.

Similarly after production and purification of soluble oligomeric TNF receptors, their analogs or derivatives can then be modified by standard chemical means for the formation of salts and functional derivatives for the manufacture of pharmaceutical compositions containing TNF-receptors of the present invention as active ingredients.

For producing a soluble, oligomeric TNF-receptors of the present invention from existing clones full receptor TNF receive a DNA sequence encoding the extracellular domain of the TNF receptor, and thus obtain the DNA sequence encoding the intracellular domain, or the "domain of death" of the TNF receptor and the intracellular domain or domain of death receptor FAS/APO1 (see Example 2 and Example 5). Then the DNA sequence must have the th extracellular domain are ligated with the DNA sequence of the desired intracellular domain, or parts of it, contains a "death domain", and thus obtained a hybrid product built (and are ligated in the corresponding expressing vector under the control of a promoter or other regulatory expression sequence. After creating the expressing vector, this vector is introduced into appropriate cells of the host (i.e. these cells transform, transferout, etc. this vector), and after expression of the specified vector in these cells receive sireny product of the present invention, which is a soluble samoassotsiiruyutsya molecule TNF receptor. Then produced thus molecules isolated from the host cells using standard techniques, and is obtained as the final product is soluble oligomeric TNF receptors.

In a preferred embodiment, the hybrid sequence encoding the extracellular domain and intracellular domain, or parts thereof, produced using the RSD-methods using oligonucleotides specific for a given target sequences copied from clones encoding the complete TNF-receptor molecule. You can use another method, which is to highlight areas that encode the extracellular domain and intracellular domain through restrictively endonucleases and subsequent splicing of known SP is way to join them, without modification or with modification of the ends of the restriction fragments to guarantee the correctness of the ligating the fragments of the receptor (i.e. extracellular and intracellular its domains or parts thereof). Thus obtained hybrid products are then inserted into the selected expression vector.

Similarly, the present invention also relates to a soluble, oligomeric FAS/APO1 (FAS)receptors containing the extracellular domain of FAS/APO1 receptor and samoassotsiiruyutsya intracellular domain of the P55-R (R-1C) its "domain of death" (55DD or samoassotsiiruyutsya intracellular domain of FAS/APO1 receptor (FAS-1C) or "domain of death" (FASDD), or any of their analogues, or derivatives (see above). Below in Example 5 provides a detailed description of the design of these soluble oligomeric FAS-receptor, where the source material used existing cloned full sequence encoding the receptor FAS and the appropriate oligonucleotides for PCR-producing the desired extracellular and intracellular domains with their subsequent legirovaniem for hybrid product, which was then embedded in need expressing vector. As described in detail above and below, for producing the desired soluble oligomeric FAS-receptors can be used as prokaryotic and eukaryotic the notches of the masters, and after the production of these receptors, they can be cleaned and used as active ingredients in pharmaceutical compositions.

The above soluble oligomeric FAS-receptors of the present invention are designed to efficiently blocking FAS-ligand, which may also exist as a trimer (similar to TNF, see above), each of the oligomeric receptor of the present invention is able to communicate with two or more FAS-ligands, and thereby neutralize their activity. It is known that FAS-ligand is mainly ligand associated with the cell surface, but it can also exist in a soluble form. In any case, oligomeric FAS-receptors of the present invention is able to contact at least two monomers of this ligand, and thereby promote more effective than Monomeric FAS-receptors) neutralizing activity of FAS-ligand. FAS-ligand, and hence activation of the FAS-receptor plays a significant role in the emergence of a number of pathological conditions, particularly conditions associated with liver damage (for example, apoptosis of hepatocytes), including liver damage associated with hepatitis, and autoimmune conditions, including destruction (apoptosis) of lymphocytes in HIV-infected patients (see, for example, Ogasawara and others 1993; Cheng. and others, 1994). Therefore, soluble oligomeric FAS-receptors of the present invention are designed to block the activity of FAS-ligand, and can be used as active ingredient in pharmaceutical compositions for the treatment of such FAS-ligand-associated pathological conditions.

Similarly, the present invention also relates to soluble oligomeric receptors with affinity binding of both TNF and FAS-R ligand, and represents the so-called "mixed" oligomeric receptors TNF-R/FAS-R. These mixed oligomeric receptors contain at least one extracellular domain of the TNF-R, and at least one extracellular domain of FAS-R, which are combined in the oligomeric receptor due to the fact that each of them Prigogine to one of the above samoassotsiiruyutsya RS, P55-DD, FAS1C or PASDD.

These mixed oligomeric receptors can be obtained by: a) the production of any of the above hybrid products that contain the extracellular domain of the receptor TNF-R p75-TNF-R or preferably p55-TNF-R), sewn with any one of samoassotsiiruyutsya domains of the P55-1C and FAS-1C, or any one of samoassotsiiruyutsya "domain of death" 55DD and FASDD, or any samoassotsiiruyutsya part, an analogue or derivative; (b) the production of any of the above hybrid p is the FL, that contain the extracellular domain of FAS-R, custom made with any one of samoassotsiiruyutsya RS: FAS-1C, p55DD and FASDD, or any part thereof, analogs, or derivatives; and (C) mixing any of the TNF-specific hybrid products (a) with any of the FAS-R ligand-specific hybrid products (b) and obtain (after standard sampling procedures and treatment) oligomeric (dimers or oligomers of higher order) receptors containing at least both of the extracellular domain of TNF-R and FAS-R, which are connected among themselves due to their ability to self-stitched with them areas 1C or DD.

For the above mixed oligomeric receptors can be used and another method, which is co-transformation of suitable host cells expressing the above vectors, one of which encodes the TNF-specific FAS-R-hybrid products, and the other encodes FAS-R ligand-specific FAS-R-hybrid products. After the expression of these different hybrid products in cells masters mixed oligomeric (TNF-R or FAS-R) receptors can be obtained by standard cleaning procedures and selection.

The value of these mixed in their affinity oligomeric receptors consists, primarily, in that they neutralize both TNF and FAS-R ligand, if there is an excessive endogenous ex who ressia, or if after exogenous introduction, they Fightstar in too large quantities. Recent observations indicate the probability that between FAS-R ligand (usually associated with the cell surface) and TNF-α (which can also be associated with cell surface) there is a synergy. Therefore, in some cases it is desirable to neutralize both of these ligands in the same place on the cell surface, i.e. so that the receptor with mixed affinity could block the binding of TNF with its receptor and the binding of FAS-R ligand with its receptor at the same time. In accordance with these receptors with mixed affinato can be used as an active ingredient in compositions intended for the treatment of these conditions (see above)in which the activity of TNF and FAS-R ligand is undesirable.

Similarly, in accordance with the above description relating to soluble oligomeric TNF-R and FAS-R, and mixed TNF-R or FAS-R - oligomers of the present invention, it is also possible to produce soluble oligomeric receptors of other species or mixed receptors originating from other types of receptors, and in particular, from any other members of the superfamily of TNF/NGF. In this case, any extracellular domains of different receptors can be p is hoedeman to the above samoassotsiiruyutsya intracellular domains, or their fragments, or any other extracellular domains of members of the superfamily, which also have the ability to self.

Expression of any of the above-mentioned protein of the present invention can be carried out in eukaryotic cells (e.g. yeast, insect or mammalian) using appropriate expressing vectors. For these purposes may be used any of known methods.

For example, DNA molecules encoding proteins obtained as described above, can be embedded in a specially constructed expressing the vectors in accordance with standard techniques (see Sambrook and others, 1989). Double-stranded DNA are ligated with plasmid vectors by homopolymer extension chain, or by restriction of stitching using synthetic DNA linkers or equipment by ligating blunt ends. For ligating DNA molecules using DNA ligase, and undesirable portions connections eliminate by treatment with alkaline phosphatase.

To implement the expression of the desired protein expressing vector should also contain specific nucleotide sequences that are capable of regulating transcription and translation, and which prisoedinyat to DNA that encodes a desired protein, so that could be the expression of CGUs is as followed produced the desired protein. To this gene could be transcribed, first of all, it is essential that this gene was preceded by a promoter that is recognized by RNA polymerase and with which the polymerase binds, initiating thus the transcription process. There are various promoters, which operate with different levels of effectiveness (strong and weak promoters). For prokarioticheskih and eukaryotic cells use different promoters.

Promoters that can be used for the purposes of the present invention are either constitutive (unregulated) promoters such as the promoter int bacteriophage Δ, the bla promoter of the gene β-lactamase RvR, and the CAT promoter of the gene chloramphenicolchloramphenicol RRR, etc. or inducible promoters, such as prokarioticheskie promoters, including the major right and left promoters of bacteriophage Δ (RLand RR), the promoters trp, recA, lacZ, lacJ, ompF, and gal, E. coli, or the trp-lac hybrid promoter, etc. (Glick V.R. (1987)). To achieve high levels of gene expression in prokaryotic cells, in addition to using strong promoters, ensuring the production of high quantities of mRNA, it is also necessary to use the binding sites with the ribosome to ensure efficient translation of mRNA. As example can serve as the Shine-dalgarno sequence (consecutive is required SD), anticipating the initiating codon and complementary to the 3' - end sequence of the 16S-RNA.

For eukaryotic hosts can be used in a variety of transcriptional and translational regulatory sequences, depending on the nature of the owner. These sequences can occur from viral sources, such as adenovirus, papilloma virus, vaccinia, simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. As examples may serve the TK promoter of herpes virus early promoter of the virus SV 40 promoter of yeast gene da and other Regulatory sequences of transcription initiation can be selected so that they are enabled to carry out the repression and activation of gene expression for modulation.

A DNA molecule having a nucleotide sequence encoding a hybrid protein of the present invention, is introduced into a vector with accordingly prisoedinenie transcriptional and translational regulatory sequences capable of integrating the desired gene sequences into cells of the host. Cells that were stably transformed introduced DNA can be selected by introducing one or more markers that allow selection of host cells that contain the ASIC expressing this vector. The marker may provide phototrophy auxotrophic hosts, resistance to biocide, for example, to antibiotics or heavy metals, such as copper, etc. of Breeding gene marker can be either directly to Prigogine to expressed gene DNA sequence, or introduced into the same cell by co-transfection. For optimal protein synthesis of the present invention can be also introduced additional elements. Such elements include transcriptional promoters, enhancers, and signal termination. Vectors for the expression of cDNA introducing these elements, described Okayama H. (1983).

In a preferred embodiment of the present invention introduced DNA-molecule is inserted into a plasmid or viral vector capable of Autonomous replication in a cell of the recipient. When selecting a particular plasmid or viral vector of the important factors are: ease of recognition and selection of cells-recipients containing the vector, cells that do not contain this vector; the number of copies of the vector that you want to get in this particular host; and the fact, whether desirable to use a Shuttle vector that can replicate in the cells of the owners of different types.

Preferred prokaryotic vectors include plasmids, such as plasmids, capable of aplitsiruetsya in E. coli such as RvR, Co1E1, puC101, rasus 184, and the like (see Maniatis and others), 1982; Sambrook and others, 1989); plasmids bacilli (Bacillus), such as RS, RS, RT, etc. (Gryczan So 1982); plasmid Streptomyces, including p1J101 (Kendall, K.J. and others, 1987); Streptomyces bacteriophages such as ⊘A31 (Chater, K.F., and others, in: Sixth International Simposium on Actinomycetales Biology, (1986); and plasmids of Pseudomonas, (John, J.F and others, 1986; and Izaki, K., 1978). Preferred eukaryotic plasmids are BPV, vaccinia virus, Sv40, 2-micron ring and the like, or their derivatives. These plasmids are well known in the art (Botstein D. and others, 1982; Broach J.R. &in: The Molecular Biology of the Yeast Saccharomyces; Life Cede and Inheritance (1981); Broach J.R. (1982); Bollon D.P. et al., (1980); T. Maniatis, In: Cell Biology: A Comprehensive Treatise. Vol.3; Gene Expression, (1980); u Sambrook et al., 1989).

After receiving the vector or DNA sequence containing the construct designed for expression, the DNA construct can be introduced into the appropriate cell host any suitable means, for example, by transformation, transfection, conjugation, fusion of protoplasts, electroporation, precipitation of calcium phosphate, direct microinjection, etc

Cell owners used in the present invention may be prokaryotic or eukaryotic. Preferred prokaryotic hosts include bacteria such as E. coli, a Streptomycete, Found Salmonella, Serrata, etc. the Most preferred use is mioticescimi hosts are E. coli. From bacterial hosts of particular interest are the strain E. coli K12 294 (ATSS 31446), E. coli. X1776 (ATSS 31537), E. coli W3110 (F-lambda-, prototrophic ADS 27325)), and other enterobacteria such as Salmonella typhimurium or Serratis marcescens, and various Pseudomonas species. B such conditions, the protein is not glycosylated. Prokaryotic hosts must be compatible with the replicon and regulatory sequences in expressing plasmid.

Preferred eukaryotic hosts are mammalian cells such as human cells, monkeys, mice, and cells of the ovary of the Chinese hamster, because they provide post-translational modification of protein molecules including correct installation or glycosylation at correct sites. Yeast cells are also capable of post-translational modification of peptides, including glycosylation. For production of the desired proteins in yeast cells, there are a number of methods using recombinant DNA technology, using strong promoters and vysokoopasnye plasmids. Yeast cells recognizes leader sequences on cloned mammalian gene products and secrete peptides bearing leader sequences (i.e. pre-peptides).

After the introduction of the vector of cell owners cultured in the selective medium used on the I selection on growth vectorstring cells. Expression of the cloned gene sequence leads to the production of the desired proteins.

Purification of recombinant proteins carry out any of the known methods commonly used for this purpose, for example by extraction, precipitation, chromatography, electrophoresis or the like, the Preferred method of purification protein of the present invention is affinity chromatography using monoclonal antibodies against TNF-receptor immobilized on a gel matrix contained within a column. For this purpose the crude preparations containing recombinant protein was passed through the column. The protein binds to the column using specific antibodies, whereas untreated proteins will pass through the column. After washing, protein elute from the gel by changing the pH or ionic strength.

As mentioned above, the term "salt" refers to salts formed carboxyl groups and to acid salts of prisoedinenia amino groups of protein molecules, which get well-known methods. Salts of carboxyl groups are inorganic salts, such as sodium and calcium salts, and salts formed with organic bases, for example amines, such as triethanolamine, arginine or lysine. Acid salts prisoedinenia are, for example, salts of mineral acids and salts of organic CI the lot.

The term "functional derivative"used in the present description, refers to a derivative, which can be obtained by modifying functional groups, fightstyle as side chains on the amino acid residues or the N - or C-terminal groups, by methods well known in the art, all such derivatives are included in the scope of the present invention provided that they are pharmaceutically acceptable, i.e. they preserve the activity of the original protein and do not confer toxic properties on compositions containing them. Such derivatives are aliphatic esters or amides of the carboxyl groups, and N-acyl derivatives of free amino groups, or O-acyl derivatives of free hydroxyl groups formed by acyl groups (for example, alkanoyloxy or carbocyclic arolina groups).

The term "fraction"used in the present description, refers to any part or fragment of the receptor (intracellular or extracellular domain), or protein, to bind the intracellular domain of the receptor, where said parts or fragments retain the biological activity of this receptor or protein.

As mentioned above, the present invention also relates to various pharmaceutical compositions containing pharmaceutically acceptable Eitel and various of the above-described active ingredients of the present invention or their salts, functional derivatives, or any of their mixtures. These compositions can be used in any of the conditions described in this application, for example, in terms of overproduction endogenous TNF, for example, in septic shock, cachexia, rejection reactions by type of graft versus host disease, autoimmune diseases such as rheumatoid arthritis, etc. route of administration of the compositions of the present invention may be by any method acceptable to the introduction of such agents, and depends on the purpose and conditions for introduction of, for example, if the composition is intended for inhibiting the action of TNF, 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 (e.g., the knee), or continuously by infusion, etc. of the Composition of the present invention can also be used in the case of TNF-intoxication caused by exogenous introduction of too high amounts (overdose) TNF, for example, anticancer or antiviral therapy.

The pharmaceutical compositions of the present invention is obtained by mixing the protein or its derivatives with physiologically acceptable carriers, stabilizers and fillers, and are made in the form of a dosage form, for example, by freeze-drying in the pharmaceutical what is a mini-bottles. The amount of active compound required for the introduction depends on the method of administration, the particular disease, and the patient's condition. For example, local injection of inflammation in the case of rheumatoid arthritis will require a smaller amount of the active ingredient on body weight than an intravenous infusion in the case of septic shock.

Other aspects of the present invention will be apparent from the following examples.

In more detail, the present invention is set forth in the following examples, not limiting, however, the scope of the present invention, and illustrated by the accompanying filocamo.

Example 1

Cloning and isolation of proteins that bind to the intracellular domains of the receptors P55 and P75 TNF

Separation of proteins interacting with the intracellular domains of the P55 and P75 TNF receptors (RS and R75 1C), was used yeast dvuhserijnaya system (Fields &Song, 1989). This dvuhserijnaya system is a genetic analysis using yeast cells, carried out for the detection of specific interactions protein-protein in vivo by restoring eukaryotic transcriptional activators such as GAL4, having two separate domains: the DNA-binding domain and activation domain"; and when the expression of these domains and their binding to each other, obrazu the Xia preparirovannyh protein GA L 4, with the ability to communicate with the upstream activating sequence, which, in turn, activates the promoter controlling the expression "reporter" gene, such as lacZ or H1S3, and this reporter gene expression can be readily observed in cultured cells. In this analysis the genes for candidate interacting proteins cloned in individual expressing vectors. In one expressing the vector sequence of one protein candidate cloned in the corresponding phase sequence DNA-binding domain of GAL4, which received a hybrid protein with the DNA binding domain of GAL4, and the other vector sequence of the second protein candidate cloned in the same phase with the sequence of the activation domain GAL4, which received a hybrid protein with the activation domain GAL4. Then these dvuhkabinnye vectors are co-transformed into yeast strain having a "reporter" gene lacZ or H1S3, adjustable upstream binding sites GAL 4. In the result, only transformed cell hosts (cotransformation), which expressed two hybrid protein is able to communicate with each other, will Express the reporter gene. In the case of reporter gene lacZ, the cells of the host expressing this gene will be acrasia the change in blue color when added to a culture X-qal. Therefore, the blue color of the colonies would indicate that the two cloned protein candidate is able to communicate with each other.

Using the specified twohybrid system, the intracellular domains RS and RS cloned separately into the vector pGBT9 (bearing DNA-binding sequence GAL 4; supplied by the company CLONTECH, USA, see below), resulting in the received hybrid proteins with the DNA-binding domain GAL 4 (similarly intracellular domain PAS-1C and part RS, namely 55DD were also cloned into pGBT9 and used to highlight other 1C-binding protein, see Example 3 below). For cloning RS and RS in pGBT9 were used clones encoding the full-size cDNA sequences p55-TNF-R (Schall and others, 1990) and p75-TNF-R (Smith and others, 1990), from which were cut intracellular domains in the following way: with the help of enzymes EcoRl and Sall cut RS, then EcoRl-Sall fragment containing the sequence RS, was isolated by standard method and inserted into the vector, restrictively, in the multiple cloning site (MCS), enzymes EcoRl and Sall. RS cut out using enzymes BspH1 and Sall, then BSpH1 - Sall fragment containing RS sequence, was isolated by standard methods, and built up a fragment of maple, resulting in a received fragment, which was inserted into the vector pGBT9, restricter the cell enzyme Smal and Sall.

Then the above-described hybrid (chimeric) vectors Ko was transferrable (separately, one co-transfection was performed using RS-hybrid vector, and using R75-hybrid vector) together cDNA library from HeLa cells humans, cloned in the vector pGAD GH bearing activation domain GAL4, a yeast strain F7 (all of the above vectors pGBT9 and pGAD GH carrying cDNA library of HeLa cells and yeast strain was obtained from Clontech Laboratories, Inc., USA, as part of MATCHHMAKER Two-Hybrid System, # PT1265-1). Co-transfected yeast were selected on their ability to grow in medium containing histidine (His--Wednesday), with growing colonies indicated the positive transformants. Then selected yeast clones were analyzed for their ability to Express the lacZ gene, i.e. on their LACZ activity by adding X-gal into the culture medium, which undergoes catabolism to painted blue product involving β-galactosidase, the enzyme encoded by the gene lacZ. Thus, the appearance of blue colonies indicates fightstyle active gene lacZ. In order lacZ gene was active, it is necessary that in the transformed clones activator GAL4 transcription was Fightstar in the active form, namely, that the DNA-binding domain of GAL4, encoded by one of the above hybrid vectors, was PR the Vilna Prigogine to the domain activation GAL 4, coded other hybrid vector. This combination is possible only in the case when two proteins connected with each of the GAL4 domains, capable of stable interaction (binding) with each other. Thus, His+and blue (LACZ+) colonies that were selected are colonies, co-transfected with a vector coding for RS, and a vector coding for a protein product derived from HeLa cells humans, and can stably contact RS, or colonies transfected with a vector coding for RS, and a vector coding for the protein product of HeLa cells capable of stably contact hrs.

From the above yeast His+, LACZ+colonies were isolated and plasmid DNA was introduced by electroporation into the strain NW E.coli in accordance with standard techniques, and then made the selection Leu+- and ampicillin-resistant transformants, i.e. transformants carrying the hybrid vector pGADCGH, which contains AmpR- and Leu2-coding sequences. So the transformants are clones carrying sequences encoding the identified new proteins that are able to communicate with RS and RS. Then from these transformed E. coli were isolated plasmid DNA and they were re-tested by:

a) re-transformation of these is one of the original hybrid plasmid, containing intracellular domain (hybrid plasmid G9 bearing sequence RS or P75-1C) by embedding in a yeast strain NR, as described above. As a control for co-transformation of plasmids encoding RS-binding protein or RS-binding protein, used vectors carrying sequences carrying irrelevant protein, for example, age-Lamin one or pGBT9. Then co-transformed yeast were tested for growth on one of His-environment, or an environment with different levels of 3-aminotriazole; and

C) re-transformation with plasmid DNA or the original hybrid plasmid and a control plasmid, described in (a), in a yeast cell host strain SF526, and then determine LACZ+activity (efficiency β-gal, i.e. the development of blue color).

The results of the above tests showed that the nature of growth of colonies in HLs-environment is identical to the nature LACZ activity, as evidenced by the colour of the colonies, i.e. His+colonies were also LACZ+. In addition, LACZ activity in liquid culture (preferred culture conditions) were assessed after transfection hybrids containing the DNA-binding domain and activation domain GAL4 in yeast cells & RU that have the best LACZ-inducibility activator GAL4 transcription than cells Hosea is and yeast strain HF-7.

The results of the implementation of the above co-transpency presented below in Table 1, which shows that the detected proteins are able to bind with RS or RS, namely the proteins identified 55.1.1 that are associated with RS; and 75.3 and 75.16 that are associated with RS. All these RS and RS-binding proteins proteins are authentic person, encoded by cDNA sequences derived from cDNA libraries of HeLa cells, and sewn with the sequence of the activation domain GAL4 plasmid pGAD GH in the above-described yeast twohybrid analysis system.

Interestingly, it was also discovered that the fragments RS, namely the proteins identified 55.1 and 55.3 able to contact RS. This fact is also discussed below in Example 2.

Table 1
Total harakteristiki some cDNA clones (see also Example 3), the selected method using twohybrid system
The hybrid containing the DNA-binding domainThe hybrid containing the activation domainThe coloration of coloniesLac Z activity analysis using liquid culture
pGBT9-1C55-white0,00
pGBT9-1C5555,1 blue0,65
pGBT9-1C5555,3blue0,04
-55,1white0,00
-55,3white0,00
Rast-Lamin55,1white0,00
Rast-Lamin55,3white0,00
pGBT955,1white0,00
pGBT955,3white0,00
pGBT9-S55,11bluenot ODA.
-55,11whitenot ODA.
Rast-Lamin55,11whitenot ODA.
pGBT955,11whitenot ODA.
pGBT9-1C7575,3bluenot ODA.
pGBT9-1C75-whitenot ODA.
-75,3whitenot ODA.
Rast-Lamin75,3whitenot ODA.
pGBT975,3whitenot ODA.
pGBT9-S75,16th is of Ubayy not ODA.
-75,16whitenot ODA.
Rast-Lamin75,16whitenot ODA.
pGBT975,16whitenot ODA.

In the above Table presents the following plasmids and hybrid encoding the DNA-binding domain of GAL4 and the domain of the GAL4 activation:

Hybrids containing the DNA-binding domain:

pGBT9-S: full-intracellular domain of the P55 TNF-R (RS)

Rast-Lamin: irrelevant protein Lamin

PGBT9: one vector

pGBT9-S: full-intracellular domain of the P75 TNF-R (p751C)

The hybrid containing the activation domain;

55.1 and 55.3 correspond to fragments of the intracellular domain of the p55-TNF-R.

55.11: a new protein associated with the P55-TNF-R

75.3 and 75.16: new proteins associated with the p75-TNF-R.

The above cloned cDNA encoding a new RS and RS-binding proteins, namely 55.11, 75.3 and 75.16 were then sequenced using the standard method of DNA sequencing. Partial sequences of all of these sequences encoding a protein shown in figa, where figure 1(a) shows the cDNA sequence encoding a protein 55.11; figure 1(b) shows the partial cDNA sequence encoding a protein 75.3; and Fig. (C) shows the partial PEFC is the cDNA sequence, encoding a protein 75.16. Figure 1(d) shows the amino acid sequence of the protein 55.11, derived from the nucleotide sequence depicted in figure 1(a).

However, it should be noted that the sequence of the cDNA that encodes a protein 55.11, it was also reported Khan. and others (1992) in their studies on cDNA sequences of the human brain, and aimed at the development of a new fast and accurate method of sequencing and physical and genetic mapping of the cDNA of the human brain. However, Khan and others say nothing about the functions or any other properties of the proteins encoded by 55.11-cDNA sequence, such as functional and other tests were not the purpose of this study Khan and his staff.

Analysis and characterization of protein 55.11

a) General methods and materials

i ) Cloning of cDNA 55.11

After the analysis (e.g., Northern analysis, see below) cDNA protein 55.11 it was found that the above-mentioned cDNA protein 55.11 cloned twohybrid screening method, represents only a partial cDNA protein 55.11 having the nucleotide 925-2863 (see figure 1(a)), which encode amino acids 309-900 (see figure 1(d)). The rest of 55.11-cDNA (nucleotides 1-924 (figure 1(a), which encode amino acids 1-308 (figure 1(d)) was obtained by standard methods, namely by PCR-cloning of the cDNA-bi is liteky fetal human liver (for more details see below). The complete nucleotide sequence 55.11 (figa)) was determined in both directions by means of dideoxy-termination circuit.

ii) Dvuhserijnyj analysis method on the expression β-galactosidase

The test expression β-galactosidase activity was performed as described above, except that instead of vector pGAD-GH containing the activation domain G14 used vector pVP16, which included activation domain VP16. The numbering of residues in the proteins encoded by cDNA-inserts, was the same as in the data Bank pGAD-GH. Deletion mutations were produced using PCR, and point mutations were produced by using oligonucleotide-directed mutagenesis (Kunkel, 1994).

iii) Northern analysis

Total RNA was isolated using R1 REAGENT (Molecular Research Center, Inc., Cincinnati, Oh., USA.) then denaturiruet in formaldehyde/formamide buffer, subjected to electrophoresis on an agarose/formaldehyde gel and blocked on the membrane CeneScreen Plus (Dupont, Wilmington, DE., USA) in 10x SS RE-buffer using standard techniques. The blots were hybridisable partial cDNA protein 55.11 (see above, nucleotides 925-2863), were subjected to radioactive tagging using set for randomized priming (Bcehringer Mannheim Biochemica, Mannheim, Germany) and washed in harsh environments. The autoradiography was carried out for 1 week.

iv) Expression of cDNA protein 55.11 in HeLa cells and binding of the s protein 55.11 protein, delivering a hybrid glutathione-S-transferase with the P55-1C

Received hybrids glutathione-S-transferase (GST) with the P55-1C (GST-p551C) and RS, truncated below 345 amino acids (GST-p551C345), and adsorbing on glutathione-agarose beads, as described below in Example 2 (see also Smith & Corcoran, 1994; Frangioni & Neel, 1993). cDNA protein 55.11 (nucleotides 1-2863, i.e., full-size cDNA 55.11), FLAG-55.11 and luciferase expressed in HeLa cells. FLAG-55.11 represent a fragment of the protein 55.11 extending between amino acid residues 309 and 900 (partial cDNA sequence of the protein 55.11 (nucleotides 925-2863), originally cloned by the method of twohybrid screening), and N - stitched with oktapeptidom FLAG (Eastman Kodak, New Haven, ct. U.S..). The expression of the hybrid proteins was performed using the tetracycline-regulated expressing vector (HtTA-1) clone of HeLa cells, which expresses the tetracycline-regulated transactivator (see below). Example 2, and Gossen &Bujard, 1992). Metabolic labelling downregulation of proteins with [35S] Met and [35S] Cys (Dupont, Wilmington, De. USA; Amersham, Buckinghamshire, England ), lysis of HeLa cells, immunoprecipitation, and the binding of labeled proteins with GST-hybrid proteins was carried out as described below (Example 2), except that the buffer for lysis of cells instead of 0.1% Nonidet P-40 was Fightstar of 0.5% Nonidet P-40. Immunoprecipitation 55.11. and FLAG-55.11 conducted with the use of what Finance rabbit antisera (diluted 1:500), produced against GST-hybrid protein containing the region 55.11 extending between amino acids 309 and 900, and mouse monoclonal antibodies against oktapeptid FLAG (M2; Eastman Kodak; 5 µg/ml of cell lysate).

a) Binding protein 55.11 with the P55-1C in the transformed yeast

This study was undertaken to identify the nature of the binding between 55.11 and RS, and in particular to identify regions of these proteins are involved in binding. This was applied dvuhserijnaya method described above, where various full and deletion mutants RS (see also below. Example 2) in the construction of "DNA-binding domain were used as "bait" to associate with "prey", which is an incomplete protein 55.11 encoded in the structures in which this incomplete 55.11 sequence (residues 309-900 initially allocated) was prigoginei "domain activation vectors GAL4AD and VP16AD. In addition, they constructed various deletion mutants 55.11, which were legirovanyh with the "activation domain" in the vector GAL4AD (for example, mutants 55.11, with only the remnants of 309-680 and 457-900). Linking different designs with the "binding domain" with different designs with the "activation domain" was investigated in transfected yeast cells SF526. Binding was assessed by analyzing the expression β-Gal is chaidez, conducted twohybrid method using a membrane filter. Irrelevant protein SNF1 and SNF4 served as control for structures with "binding domain" and "domain activation", respectively; "empty" vectors (i.e., not containing Ga14 (pGAD-CH) and VP16 (V16) served as a negative control for structures with "activation domain"; and "empty" G14-vector (pGBT9) served as a negative control for structures with "binding domain". The results of the analysis are presented below in Table 2, where the characters "+++" and "++" indicate the development of a bright color within 20-60 minutes after the start of the analysis, respectively (positive link); and the symbol "-" indicates no staining 24 hours after the start of the analysis (negative results). A blank space in Table 2 means that the analysis of the binding did not).

From the results shown in the above Table 2, we can conclude that 55.11 associated with the P55-1C in the website, which is not the "death domain" (residues 328-426) P55-1C.

Protein 55.11 was associated with shortened P55-1C, which was deleterows "domain of death" (design 206-328 in Table 2), more efficiently than the full P55-1C. In addition, this protein even contacted more shortened at the C-end P55-1C (design 206-308), and with constructie is, has been deleted "domain of death" and the membrane-proximal part of the P55-10 (design 243-328). However, protein 55.11 not been associated with the design, which was shortened by the N-end amino acids 266 (table 2). These data suggest that the binding site for 55.11 located in the area stretching between residues 243 and 308 P55-1C, and that N is the end of this binding site is located between residues 243 and 266.

The transfer of the cDNA for the protein 55.11 cloned from the original design "production", which contained the activation domain GaL4, in design "production", containing the activation domain P16 did not reduce the efficiency of binding protein 55.11 with the P55-1C (table 2). Thus, the structure (or structures)that are involved in this binding, obviously, is the molecule 55.11 and do not include site merge 55.11 domain activation.

However, binding 55.11 with the P55-1C does not occur even when limited truncation of the protein 55.11 in it (55.11-design 309-680)-end N-end (55.11-design 457-900). (The remainder of the 309 is the first residue in the protein 55.11 encoded by the partial cDNA clone initially allocated at twohybrid screening).

The observed link between 55.11 and the P55-1C is clear, specific, because 55.11 not been associated with other proteins, including three receptor family, receptors of the TNF/NGF (P75-R, FAS/APO1, and CD40), and proteins such as l the min and cyclin % (data not shown). It should be noted that other analyzed TNF/NGF-receptor proteins were also tested those parts of them that contained the intracellular domain of FAS-R man (residues 175-319), CD40 (residues 216-277) and p75-TNF-R (residues 287-461), and none of them contacted 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-55.11-cDNA

The investigated cell lines were HeLa cells, CEM, Jurkat and G2 originating from epithelial carcinoma human acute lymphoblastic T-cell leukemia, acute T-cell leukemia, and hepatocellular carcinoma, respectively. First dedicated 55.11-cDNA (nucleotides 925-2863) was used as probe. Samples consisted of 10 µg RNA per track. The results of the Northern blot analysis shown in figure 2, which is reproduced Northern blot.

Thus, from figure 2 it is seen that the result of Northern blot analysis carried out for several cell lines using 55.11-cDNA as a probe identified a single hybridities transcript of approximately 3 kb, which was larger than the cDNA (2 b) from the originally selected 55.11-cDNA. Using oligonucleotide primers that corresponded to the sequence 55.11, we cloned using PCR, 5'-extending the sequence, DL is on which was 1 kb. The sum of the lengths of the 5'-extending section and initially cloned cDNA sequences approximately equal to the length of this 55.11-transcript. 3 kb cDNA, which included both of these parts, effectively expressives in HeLa cells (see below) with the formation of a protein of approximately 84 kDa, suggesting that this 3 KB-cDNA contains the site of translation initiation.

d) In vitro binding protein 55.11 with GST-hybrid proteins containing region P55-1C

In order to ensure that 55.11 can really connect with the P55-1C, and that the yeast proteins are not involved in this binding was investigated in vitro the interaction of GST-P55-1C-hybrid proteins produced by bacteria, protein, coded 3 kb - 55.11 - cDNA (full-size 55.11), and produced transfected HeLa cells. In this study, cDNA for full-length protein 55.11, FLAG-55.11 (residues 309-900 protein 55.11 encoded originally cloned partial cDNA, and merged, in its N end, with oktapeptidom FLAG), and luciferase (control) expressed in transfected HeLa cells, and were metabolically labeled with [35S] Met and [35S] Cys. With GST were hybridized following proteins: full-P55-1C (GST-P55-1C) and P55-1C, truncated at the C-end amino acids 345 (GsT-p55-1C345) in order to remove the "domain of death" (see Table 2). One GST served as the counter is La. Lysates of transfected cells were subjected to thus with antibodies against protein 55.11 in if you want to associate with GST-hybrid proteins used full size 55.11; or with antibodies against oktapeptid PLAG in if you want to associate with GST-hybrid proteins used FLAG-55.11-a hybrid product. Proteins were analyzed by electrophoresis on polyacrylamide gel with LTO (LTOs-PAG: 10% acrylamide), and then subjected autoradiography.

On figa and 3B presents autoradiogram above LTO-SDS page gels, where figa illustrates the binding of the full-size protein 55.11 (55.11-full) with different GST-hybrid proteins; and figv illustrates the binding of Flag-55.11-hybrid product with various GST-hybrid proteins. On figa, on the outermost track to the right, shows the control immunoprecipitate lysates of cells transfected with only one full-size 55.11 and immunoprecipitating antibodies against 55.11 (antibodies α55.11). On figv on the outermost track to the right shows the control precipitate lysates of cells transfected with only one FLAG-55.11 and immunoprecipitating antibodies against FLAG (antibodies α FLAG).

Thus, from figa and 3B shows that the protein encoded cDNA full-55.11, can be expressed in cells Not La and connect with hybrid the proteins, containing full P55-1C (GST-RS) or truncated P55-1C, which is missing most of the "domain of death" (GST-p551C345) (figa). Full-size protein 55.11 not contacted one GST (control). Similarly protein expressed in HeLa cells and the encoded originally cloned a partial cDNA for 55.11, hybridized with oktapeptidom FLAG (FLAG-55.11), was contacted in vitro with GST-p551C and GST-p551C345, but was not associated with GST (pigv). The above results are also additional evidence (see above, p.(b))that 55.11 associated with the site p551C above "domain of death", i.e. in the region of the P55-1C, which is located closer to the transmembrane domain.

In addition, the above study also showed that, in accordance with the present invention can be successfully produced antibodies against 55.11 (figa).

e) Comparison of the derived amino acid sequence of the protein 55.11 person with sequences of related proteins, fightstyle in lower organisms, and particularly the sequence of the protein 55.11

As mentioned above, in accordance with the present invention was cloned and sequenced the cDNA for full-length protein 55.1 (see nucleotide sequence of figure 1(a)), and from this cDNA sequence was derived full consecutive amino acid is required 55.11 (see amino acid sequence in figure 1(d)). The study of sequences available in the data Bank (GenBankTM/ /ENG DataBank), showed that parts of the sequence 55.11-cDNA person (per. No. t, 19559 and F0128) and its murine homologue (per. No. h and 231147) were already defined in the process of random sequencing of cDNA libraries. cDNA sequence (incoming №18247)which encodes a human protein in 596 amino acids, fightstyle in cell cultures of hepatoma NC person, the same sequence that encodes a protein 55.11. However, in the protein of this hepatoma missing N-terminal part (amino acids 1-297), corresponding to that of 55.11, and in addition, this protein differs from 55.11 in the areas corresponding to residues 297-377 and balances 648-668 in 55.11. The study of sequences available in the Bank data also showed that proteins, fightstyle in Saccharomyces cerevisiae (yeast), Arabidopsis thaliana (plant Resusci tal) and Caenorhabditis elegans (worms). Thus, it is obvious that 55.11 make evolutionary conservative function. In yeast Fightstar two known protein (open reading frame UNRS and SEN3), a DNA sequence which is similar to DNA sequence 55.11. The size of both of these sequences are also close to the size 55.11. The sequence UNRS only became known by sequencing genomic CL is on, while the sequence SEN3 was cloned as cDNA. Sites in 55.11, which are similar sites in SEN3, correlate identities with sites in Wnrs, although, obviously, the major similarity is observed between 55.11 and UNRS than between 55.11 and SEN3. The available data (though only partial) of DNA sequences for proteins of Arabidopsis thaliana and Caenorhabditis elegans clearly show that these proteins are similar to 55.11 as protein UNRS yeast. Of these four proteins, the nature of which has been identified to date, only one protein, namely protein yeast SEN3 has limited homology with 55.11.SEN3 was identified as yeast equivalent subunit R activator calpaine 20S (proteolytic Central part (core) of the calpaine 26S Rechsteiner and others, 1993; DeMartino and others 1994) (..Culbertson and .Hockstrasser, personal communication).

Figure 4 schematically illustrates the comparison of the derived amino acid sequence 55.11 person with sequences of the above-mentioned related proteins, fightstyle in lower organisms. Sequences that are compared in figure 4, are the amino acid sequences predicted for: 55.11-cDNA (see figure 1(d)); the open reading frame (UNRC) in cosmides originating from the 8th chromosome Saccnaromyces cerevisiae (nucleotides 21253-24234, tolerance 10399); cDNA of the protein of saccharomyces cerevisiae (tolerance 06321); partial cDNA of the protein of the RAS is to be placed Arabidopsis thaliana (resusci tal) (tolerance T) and a partial cDNA of the protein of the nematode Caenorhabditis elegans (tolerance 27396). Figure 4 sequence "KEKE" 55.11 marked with a solid line, and the sequence AYAGS(x)8'LL dotted line. A comparative analysis of the primary structures of the sequences was performed using programs P1LEUP and PRETTYBOX of the GCG package. Gaps introduced to maximize comparison, indicated by dots.

With regard to the distinctive features of various sequences or motifs, fightstyle in the sequence 55.11 man, were made the following observations. In the protein encoded 55.11 sequence, except for the repeating sequence "CAKE", which is between Lys 614 and GLu 632 (highlighted in figure 4), "motives" conservative amino acid sequence were found. Such sequences "CAKE", which Fightstar in many proteins, including proteasome subunits and proteins "mentors" (chaperonins), which can stimulate the Association of protein complexes (Realini and others, 1994). The sequence AyAGS(x)8LL 11 appears in the sequence of the protein 55.11 twice (in sites 479 and 590, see figure 4), however, any functional role of this sequence has not been established.

f) Distinguishing features of the sequence region of the P55-1C, involved in protein binding 55.11

As described above (see PP. (b) and (d)), protein 55.11 associated with the region of the P55-1C, RAS is than necessary, between residues 243 and 308 (N is the end of this binding site is located between residues 243 and 266), and above "domain of death" and closer to the transmembrane" domain of the p55-TNF-R. This area in the P55-1C, which is associated protein 55.11, has a high content of prolinnova, serine and treoninove residues. However, this region does not contain enriched prolinnova "motives" R1 and RP2, fightstyle in some other cytokine receptors (O'neal Yulee, 1993). In the field, which extends between residues 243 and 266 and the deletion of which leads to the absence of binding of the P55-R 55.11 (see above, paragraphs (a) and (b), and table 2), two serine residues and two treoninovymi residues followed by prolinnova residues, which makes them potential Sagami for phosphorylation MAR-kinase, CD2 and other polynesienne kinases (Seqer and Krebs, 1995), phosphorylation at this site receptors can affect protein binding 55.11.

Taking into account the above data related to protein 55.11 and its binding to the P55-1C, we can conclude that, in accordance with the present invention discovered a new protein that binds to certain area, located above the "domain of death" P55-1C. This link should influence TNP-mediated activity, with the exception of inducing cell death. As shown previously, the area that is associated 55.11, participates in the induction of the nitric oxide synthase (Tartaglia and others, 1993), and, Acevi is but participates in the activation of neutral sphingomyelinase the tumor necrosis factor (Wiegmann and others, 1994). Thus, it is possible that the Association (binding) protein 55.11 with the intracellular domain of the p55-TNF-R. (p551C) affects or participates in (i) the signal transmission for the above or other TNF-effects; (ii) laying or processing of the protein (as is supposed, on the basis of similarity 55.11 with a subunit of the 26S proteasome); or ( iii) regulation of the activity or the expression of the p55-TNF-R.

Example 2

The ability to self-intracellular domain of the P55 TNF receptor (RS), its ability to cause cell death and its other distinctive features and functions; and the intracellular domain of the cognate receptors, FAS/APO1

As mentioned above, in Example 1, it was found that the intracellular domain of the P55-T G-R (RS) the ability to communicate with yourself, and in addition, it was found that the fragments RS, namely proteins 55.1 and 55.3 also able to contact RS.

It is known that binding of TNF to P55 TNF-E leads to zitotsydnoe action on cells bearing this receptor. In addition, antibodies against the extracellular domain of this receptor can stimulate this action in accordance with the efficiency of cross-stitching these receptor antibodies.

In addition, mutation research (Tartaglia and others, 1993; Brackebuch and others, 1992) showed that the function of the P55-R C is dependent on the integrity of its intracellular domain. Therefore, there is reason to believe that the initiation signal for Titorenko effect of TNF is the result of the Association of two or more extracellular domains of the P55-R (P55-1C)induced aggregation of receptors. The results obtained in accordance with the present invention, support this conclusion and indicate that the expression of the intracellular domain of the P55-R in cells without the transmembrane or intracellular domain stimulates the death of these cells. It was shown that these free intracellular domains of the P55-In capable of self, that, obviously, is the cause of their ability to operate independently of TNF. The fact that the signal transmission full-P55-R depends on the stimulation of TNF reflects, as expected, the activity of the transmembrane or extracellular domain of the receptor, which helps to reduce or prevent the self.

The ability of the intracellular domain of the P55-R (P55-1C) to self was discovered by accident when trying to clone effector proteins that interact with this receptor (see above. Example 1). For these purposes has been applied to the above "dvuhserijnyj" method. It was found that the addition of a new protein 55.11 associated (bound) with the P55-1C, and it was also found that three other cloned cDNA in HeLa cells with the hold cDNA sequences, encoding part of the intracellular domain of the P55-R, which means that the P55-1C has the ability to self. These two clones were identical and contained the insert, which encodes amino acids 328-426 (these clones were designated clone 55.1 coding for the protein fragment 55.1 RS). The third clone contained a longer insert, encoding amino acids 277-426 (this clone was designated clone 55.3 coding for the protein fragment 55.4 RS).

In addition, we assessed the in vitro interaction between the two produced in bacteria chimeras RS, one of which was hybridized with maltesewatertaxis protein (MBP), and the other was hybridized with glutathione-5-transferase (GST). These chimeras were designed, cloned and expressed using standard methods. After their expression, assessed the self-intracellular domain of the P55-R (RS) by analysing the interaction of the above bacterial produced chimeric proteins GST-1C55(Mr=51 kDa) and MBP-S (Mr=67 kDa) with each other. For this, equal amounts of GST Chimera-S (sample tracks 1-4 figure 5) or one GST (sample tracks 5-8 figure 5) immobilizovana on glutathione-agarose beads ( Sigma)and then incubated with the same amount of MBP-S-hybrid protein in one of the following buffer solutions:

i) buffer 1 (20 mm Tfig-HCl, pH 7.5, 100 mm KlC, 2 mm CaCl 2, 2 mm MgCl2, 5 mm DTT, 0.2% Triton X100, 0.5 mm PMSF, 5% Glycerol). This buffer was used for samples of tracks 1 and 5 in figure 5.

ii) buffer 1, containing 5 mm EDTA instead of MgCl2. This buffer is used for samples of tracks 2 and 6 in figure 5.

iii ) buffer 1 containing 250 mm instead of 100 mm KCl. This buffer is used for samples of tracks 3 and 7 in figure 5.

iv ) buffer 1 containing 400 mm instead of 100 mm KCl. This buffer is used for samples of tracks 4 and 8 in figure 5.

After incubation with rotation for 2 hours at 4°the beads were washed in the same buffer, and then boiled in LTO-PAG-buffer followed by electrophoresis in SDS page. Proteins on the gel were subjected to Western-batrouney on nitrocellulose membrane, which was then stained with polyclonal anticorodal against MBP. Specified stained Western blot depicted in figure 5 (samples on tracks 1-8 described above).

From figure 5 it is seen that the Chimera RS-ISI associated with the Chimera p551C-GST (lanes 1-4), regardless prihodti divalent cations, and even when a sufficiently high concentration of salt (0.4 M KCl). Hence we can conclude that RS has the ability to aveneu self.

To assess the functional role of this ability P55-1C to self, we attempted to Express the P55-1C in the cytoplasm of cells, which is susceptible to zitotsydnoe action NF. Taking into account the fact that the P55-1C, in turn, may be cytotoxic, we chose the inducible type of expression, using a newly developed strictly regulated tetracycline-controlled expressing system of a mammal (Gossen and Boujard 1992). The expression of the P55-1C resulted in massive cell death (6, right panel). The dying cells have been the loosening of the cell surface, as is the case for TNF-killing cells. Transfection of the P55-1C-structure in cells in Prilutskii tetracycline, which significantly reduces the level of expression of D10-3-regulated structures up to 105-fold reduction still results in the death of some cells, though to a much lesser extent than that observed in the absence of tetracycline (Fig.6, left panel). In contrast, cells transfected with the control construct containing the luciferase cDNA, did not detect any signs of death (results not shown).

The ability of the P55-1C to stimulate cell death, when its expression without the transmembrane or extracellular domains of the receptor, once again suggests that this domain is involved in signal transmission. In addition, this fact indicates that none of the other parts of the receptor does not play a direct role in the transmission of the signal is. Studies of the effects of mutations, including mutations of the present invention, the function of the P55-1C showed that for this function the most important area is the area that stretches between amino acid residues 326 and 407. This area reveals a noticeable similarity with sequences of the intracellular domains of the other two receptors, which are evolutionary related receptor p55-TNF, namely FAS-receptor (Itoh et, 1991; Oehm and others, 1992), which is also capable of transmitting citizeny signal; and CD40 receptor (Stamenkovic and others, 1989), which enhances cell growth; therefore, the sequence of this region, obviously, contains conservative motif, which plays a major role in the signal transmission. Since there are no similar known motifs characteristic of enzymatic activity, then, obviously, this area is signaled indirectly, i.e. perhaps it serves as a "Stikovica-receiving site for the transmitting signal of the enzymes or proteins that transmit stimulatory signals for these enzymes. All P55-1C, FAS-receptor and CD 40 can be stimulated by antibodies against the extracellular domain. This stimulation has been shown to correlate with the ability of antibodies to cross-linking receptors. Therefore, it is obvious that the signal transmission is the INIC is associated by the interaction of two or more intracellular domains, induced by aggregation of extracellular domains. Participation in this interaction of receptors with subsequent transfer of the signal was also established by research (Brackebuch and others, 1992), who showed that the expression of the receptor deprived of their functions by mutation of their intracellular domain, had a "dominant negative" effect on the function of co-expressed normal receptors. Aggregation of p55-R in response to TNF suggests that it is passive and only due to the fact that each of the molecules of TNF, which is homotrimers, can communicate with two or three receptor molecules. However, the data obtained as a result of implementation of the present invention, suggesting that this process is slightly different type.

Trend P55-1C to self-suggests that this domain plays an active role in their induced aggregation. In addition, this activity P55-1C is obviously sufficient for the initiation of signal transmission, because, when its expression independently from the rest of the receptor molecule, it can stimulate cell death in the absence of TNF or any other external stimuli. However, when its expression as the full-length receptor, this P55-TNF-R does not transmit the signal, if it is not with muliawan TNF. Therefore, we can conclude that upon activation of the P55 TNF receptor, TNF, actually overcomes some inhibiting mechanisms, warning the spontaneous Association of the intracellular domains, and this inhibition is due to binding to the P55-1C with the rest of the receptor molecule. The inhibition may be due to the orientation given to the intracellular transmembrane domain: extracellular domain; binding of some other protein with the receptor; or, more likely, a limited number of receptors that may be located in the plasma membrane. Of course, that this mechanism of regulation should be effective enough, because, according to some estimates, the binding of even only one TNP-molecule and a cell is sufficient to stimulate its destruction.

Spontaneous transmission of signals, regardless of the ligand, can lead to significant disruption of the process, regulated by this receptor. The most famous example of this is the termination of the regulation of receptor growth factor. For example, in the uncontrolled growth of tumor cells play an important role mutations that result in the initiation of signal transmission becomes spontaneous, for example, mutations that cause spontaneous aggregation of receptors. It is well known that TNP-effects, when they are excessive induction of play VA the role in the pathology of many diseases. The power of free intracellular domains (RS) the P55 TNF-receptor-independent TNF signaling may contribute to such pathological phenomena. It is possible, for example, that the cytopathic effect of some viruses and other pathogens due to their indirect cytocidal activity, and proteolytic cleavage of the intracellular domain of the P55 TNF-receptor, leading to TNF-like cytotoxic effect.

To more clearly identify the area (or areas) in RS responsible for its ability to self-and therefore for its ligand-independent cytotoxicity, and to determine whether there are other related members of the family of TNF/NGF receptors (such as FAS-R intracellular domain with the ability to self-and ligand-independent action were conducted thorough research, described below.

(a) General methods and materials

i) Dvuhserijnyj screening and dvuhserijnyj test on the expression β-galactosidase

cDNA insert encoding the P55-1C and its deletion mutants, FAS-1C and various other proteins (see Table 3) were cloned using the RIC of a full length cDNA was previously cloned in our laboratory, or from commercially purchased cDNA libraries. The expression β-galactosidase in yeast (reporter strain F526; Bartel and others, 1993), transformieren the x specified cDNA in the vector. pGBT-9 and pGAD-CH (design DNA-binding domain (DBD) and activation domain (AD), respectively) was evaluated using a test solution (guarente, 1983); and conducted analysis on filters for qualitative assessment of the same results (not shown). Dvuhserijnyj screening (Fields and Song 1989) purchased G14-A1-labeled cDNA library of HeLa cells (Clontech, Palo Alto, Ca., U.S..) for proteins that bind to the intracellular domain of the P55-R (P55-1C) was carried out using a yeast reporter strain F7 in accordance with the manufacturer's recommendations. The positivity of the selected clones was assessed by (a) prototrophic transformed yeast in respect of histidine in their cultivation in Prilutskii 5 mm 3-aminotriazole; (b) the expression β-galactosidase; and (C) test the specificity of the interaction with the SNF4 and designed, merged with Ga14 DBD).

ii) self-Association in vitro P55-1C-hybrid proteins produced in bacteria

Glutathione-S-transferase (GST) and the hybrid protein "glutathione-S-transferase - P55-1C (GST-p55-1C) was produced as described in the literature (Frangioni and Neel, 1993; Ausubel and others, 1994). Maltsevkaya (IMM) hybrid proteins were obtained using vector pMA1cR1 (New England Biolabs ) and purified on a column with amylose resin. Interaction MURR - and GST-hybrid proteins was investigated by sequential incubation glutathione-agarose beads with GT and MURR-hybrid proteins (5 μg protein/20 ál beads; the first incubation was carried out for 15 minutes and the second for 2 hours, both at 4°). Incubation with MBP-hybrid proteins was carried out in a buffer solution containing 20 mm Tfig-HCl, pH 7.5, 100 mm KCl, 2 mm CaCl2, 2 mm MgCl2, 5 mm dithiothreitol, 0,2% Triton X100, 0.5 mm phenyl-methyl-sulfonyl fluoride and 5% (by volume) glycerol; if it was necessary, in the same buffer containing 0.4 M KCl, and 5 mm EDTA instead of MgCl2. The Association MVP-hybrid proteins was assessed by electrophoresis in polyacrylamide gel with LTOs (10% acrylamide) proteins associated with the glutathione-agarose beads, and subsequent Western blotting. The blots were probed using rabbit antisera against MBP (produced in our laboratory) and goat anti-rabbit immunoglobulin conjugated to horseradish peroxidase.

iii) Induced the expression of the P55 receptor and its fragments in HeLa cells

Cells Not La, expressing the tetracycline-controlled transactivator developed by Gossen and Bujard (clone HtTA-1 (Gossen and Bujard, 1992)), were cultured in modified according to the method of Dulbecco environment Needle containing 10% fetal calf serum, 100 µg/ml penicillin, 100 μg/ml streptomycin and 0.5 mg/ml neomycin. cDNA insert encoding the P55-R or its fragments, was inserted in the tetracycline-controlled expressing vector (pUHD10-3, courtesy of H.Bujard). The cells were transferrable expression construct (5 μg DNA/6 cm Cup) by precipitation with calcium phosphate (Ausubel and others, 1994). Results transient expression of transfected proteins was evaluated at appropriate intervals after transfection in Prilutskii or in the absence of tetracycline (1 μg/ml). Cell clones stably transfected with the P55-1C-human cDNA in the vector ptHA 10-3, confirmed by cDNA transfection into cells HtTA-1 in Prilutskii tetracycline together with a plasmid carrying resistance to hygromycin, and subsequent selection of clones resistant to hygromycin (200 μg/ml). Expression of the cDNA was obtained by removal of tetracycline, which in other cases was constantly Fightstar environment for cell growth.

iv) Assessment of TNF-like effects induced induced expression of the P55-R and its fragments

The influence of induced expression of the receptor and TNF on cell viability was evaluated by the method of absorption of neutral red (Wallach, 1984). The induction of gene expression of 1L-8 was assessed using Northern blot analysis. RNA was isolated using TP1 PEAGENT (Molecular Research Center, Inc.), then denaturiruet in formaldehyde/formamide buffer, subjected to electrophoresis on an agarose/formaldehyde gel, and blocked on the membrane Gene Screen Plus (DuPont) 10 x RE-buffer IP is by the use of standard techniques. Filters were hybridisable 1L-8-cDNA probe (Matsushima and others, 1988) (nucleotide 1-392), were subjected to radioactive tagging using a set of random priming ( Bcehringer Manheim Mannheim Biochemica, Germany), and washed in harsh conditions in accordance with the manufacturer's instructions. The autoradiography was carried out within 1-2 days.

v) evaluating the expression of TNF receptor

Expression of TNF-receptor in samples 1×106cells were evaluated by measuring the binding of TNF labeled With [1251] the previously described method using chloramine-T (Holtmann and Wallac, 1987). Was also analyzed EL1SA carried out as described in the literature for the quantitative estimation of soluble TNF-receptors (Aderka and others, 1991), except that the lysis of the cells (70 μl/106CL) and for cultivation of tested samples used buffer R1 (10 mm Tfig-HCl, pH 7.5, 150 mm NaCl, 1% NP-40.1% desak-sholat, 0.1% of LTOs, and 1 mm EDTA). A soluble form of the P55-R, isolated from urine, served as a standard.

o) Mutational analysis of the intracellular domain of the p55-R (P55-1C) to identify areas P55-1C, participating in self -

As mentioned above, the P55-1C has the ability to self-and there are such areas P55-1C, which are able to communicate with full-P55-1C. In particular, was identified by one of the fragments of the P55-1C (indicated by Belk is the first fragment 55.1 in Example 1, see above), which is capable of strong binding to full-P55-1C; however, after sequencing of this fragment was determined that it is located between amino acid residues 328 and 426 the P55 TNF receptor and is the P55-1C. In addition, it was found (see below)that the above region, namely the fragment 55.1 himself capable of self, and stimulation of cytotoxic effects on the cells. Therefore, this region of the P55-1C was called the "death domain", which is located between amino acid residues 328-426 the P55-R man, and in all probability consists of amino acid residues, between about residue 328 and 414.

The fact that the "domain of death" in the P55-1C capable of self, was discovered by accident. After screening cDNA libraries in HeLa cells using twohybrid techniques (see above, Example 1) in order to identify sequences encoding proteins that are associated with the intracellular domain of this receptor, was found among the cDNA products are specifically associated with a hybrid protein "intracellular domain - GALA DBD), several clones (for example, 55.1 and 55.3), directly coding region of the intracellular domain of the P55-R (P55-1C; marked with asterisks in Table 3).

Using twohybrid test to assess the degree of specificity in self-P55-1C and Bo is precisely defined area, participating in this self, the following data were obtained (table 3): a) self-Association P55-1C restricted to the region, located in the "domain of death". Its N end is located between residues 328 and 344, and its With-the end is near residue 404 and slightly higher from the C-end (as indicated in the literature) this domain (residue 414). (b) the Deletion of the membrane proximal region of the P55-1C above "domain of death", enhances self, which suggests that this area has an inhibitory effect on the Association, (C) Murine P55-1C capable of self, as well as linking with the "death domain" of the P55-R man. d) Research self intracellular domains of the three other receptors belonging to the TNF family/GF receptors, namely receptor FAS/APO1 (FAS-R), CD40 (Fields u Song 1989) and p75-TNF receptor (Smith and others, 1990), showed that FAS-1C, which passes citizeny signal using the sequence -"motive", akin to the "death domain" of the P55-R, capable of self, and to nekotoroi degree of Association with the P55-1C. However, CD40-1C, which provides transmission of growth-stimulating signals (even though it also contains a sequence similar to the "domain of death"), and P75-1C, which is not structurally similar to the P55-1C, tend not to self, and is not associated with the P55-1C and the and FAS-1C.

In the above Table 3 illustrates the quantitative analysis of the interaction G14-hybrid structures, which includes the following proteins: intracellular domain of the human P55-R and its various deletion mutants (residues are numbered as in the work Loetscher and others (1990); murine intracellular domains of the P55-R (residues 334-454; numbered as in the work of Goodwin and others, 1991); murine FAS/APO1 (FAS 1C, residues are numbered as in the work of Watanabe - Fukanaga and others, 1992); people CD40 (CD40-1C, 216-277, residues are numbered as in, Stamenccvic and others, 1989); and people P75 TNF receptor (P75-1C, 287-461, residues are numbered as in Smcth and others, 1990). SNF1 and SANF4 were used as positive controls for the Association (Fild and Sond 1989), and Lamin was used as a negative control (Bartel and others, 1993). Proteins encoded G14-AD-structures (pGPT9), are given in a vertical column, and the proteins encoded by Ga14-A1-structures (pGAD-CH), given horizontally. Two deletion mutants marked with asterisks were cloned by twohybrid screening cDNA libraries of cells BeLa (Clontech, PalO Alto, Ca., U.S..) using the P55-1C cloned into pGBT9, as "bait". In this screening, four out of about 4×106investigated cDNA clones were positive. It was found that three of these clones correspond to the DVS plots is for the P55-R man (two identical clone, encoding residues 328-426, and one clone that encodes residues 277-426). The fourth clone, as it was found that encodes an unknown protein. Data on the expression β-galactosidase were obtained as averages of the analyses of two independent transformants, and represented as the number β-galactosidase product (unit of activity was defined as OD420×103/OP600the yeast culture, and the reaction time is given in minutes. The limit of detection in this assay was 0.05% Deviation between duplicate samples in all cases were less than 25% from the average value (not defined).

In vitro test for the interaction of bacterial hybrid protein P55-1C-glutathione - S-transferase (GST)" hybrid protein "P55-1C-maltatoday protein (MBP)affirms that the P55-R is capable of self, and excluded the participation of the yeast proteins in the binding (see above). On the specified self-Association was not inuenced neither by elevated concentrations of salt or EDTA (see above).

To assess the functional role of self "domain of death", we investigated the mechanism by which induced the expression of the P55-R or its fragments affects the cells sensitive to TNF cytotoxicity. The results of this analysis are shown in Fig.7, which shows the ligand-independent stimulation Titorenko effect in KL is located HeLa, transfected p55-R, its intracellular domain (P55-1C) or its fragments (including the "domain of death").

7 schematically shows the different DNA molecules encoding different types of TNF receptors included in the vectors, which were transfected HeLa cells (left edge Fig.7); expression (left and middle columns); as well as the viability (right column) in HeLa cells expressing various short full-size proteins p55-R, P55-1C, or fragments P55-1C, or as a control, luciferase (luc) (each of them are shown on the left side of Fig.7), while the expression was performed using the tetracycline-controlled expressing vector. Where there's no shading rectangles (left, middle and right columns) figure 7 correspond to cells transfected into Prilutskii tetracycline (1 μg/ml), which inhibits expression; and the shaded rectangles (left, middle and right columns) figure 7 correspond to the cells transfected in the absence of tetracycline. Expression of TNF-receptor was evaluated at 20 hours after transfection using ELISA, and antibody against the extracellular domain of the receptor (see the schematic illustration on the left side of Fig.7), as well as by determining the binding of radioactively labeled TNF cells (middle column). Cytocidal effect transferir the bathrooms proteins was assessed 48 hours after transfection. Data are given for 1-3 experiments with similar quantitative results, where each design used in duplicate. "ND" = " not defined".

Thus, from Fig.7. it is seen that when using the expressing vector carrying out strictly controlled expression of transfected cDNA using the tetracycline-regulated transactivator (Gossen and Bujard, 1992), a simple increase in the expression of the P55-R in HeLa cells by transient expression of transfected cDNA that encodes a full-receptor, leads to massive cell death. If expression of only one P55-1C was observed even higher cytotoxicity. A significant level of cytotoxicity was observed when the expression of only part of the P55-1C, containing mostly "death domain" (residues 328-426), in HeLa cells. On the other hand, the expression of fragments of the P55-1C, in which there is no "domain of death" or contains only a part of it (or the expression of luciferase gene used as an irrelevant control), did not exert any effect on the cell viability. Cytotoxicity P55-1C was also confirmed using cells stably transformed its cDNA. These cells continued to rise when not induced the expression of the P55-1C, but with the expression of the P55-1C, the cells were killed (see above).

(C) On the other functions of the intracellular domain of the P55 TNF-receptor

In order to determine whether stimulated other functions of TNF by self-Association of the intracellular domain, including the domain of death, we analyzed the effect of increased expression of full-length receptor (p55-R) and the expression of the intracellular domain of the receptor R-1C) on the transcription of interleukin-8 (1L-8), which is known to be activated TNF (Matsushima and others, 1988). The results of this study are presented in Fig, which is illustrated ligand-independent induction of gene expression of 1L-8 in HeLa cells, transfected with the P55-R or P55-1C using tetracyclinestrained design (see also "General methods and materials, and Example 1). Panel And Fig illustrated by Northern blot analysis (see above, Chapter "General methods and materials) RNA (7 μg/lane)extracted from HeLa cells (HT-1), untreated (control) or treated ("TNF") TNF (500 µg/ml, 4 hours), or cells HTta-1 24 hours after transfection (Prilutskii or in the absence of tetracycline) cDNA for the P55-1C ("P55-1C"), the P55-R ("The P55-R"), or luciferase ("LUC"). In the panel on Fig illustrated by Northern blot analysis, where staining was performed IS.S-pPHK methylene blue in each of the samples shown in panels And Fig.

Thus, as can be seen from Fig, transfection of HeLa cells tetracycline-controlled design, containing the P55-R-cDNA induced TRANS is iptio 1L-8. In cells transfected with a cDNA for the P55-1C, was even more pronounced induction. In both cases, the induction occurred only in the absence of tetracycline in the growth medium, suggesting that this induction is the result of the expression of transfected P55-R or P55-1C. Transfection of cells luciferase cDNA, used as control, did not exert any effect on the transcription of the gene 1L-8.

Based on the above results, it is obvious that a simple increase in the expression of p55-R, or even the expression of only its intracellular domain (P55-1C) is sufficient for inducing ligand (TNF)-independent cytotoxicity and other effects, including increased gene expression of 1L-8 in cells. The induction of these effects are likely caused by self-Association of the intracellular domain of the P55-R (P55-1C). Since, as mentioned above, it is obvious that after self-P55-1C its "domain of death"in the first place, responsible for signal transmission, it is possible that the induction of intracellular processes leading to stimulation of cytotoxicity in cells, as well as other effects, such as inducing the expression of the gene 1L-8-mediated other areas of the P55-1C after their savassaziye. Therefore, it is possible that different areas of the P55-1C responsible for choosing the e TNF-induced effects (e.g., cytotoxicity and induction of gene expression of 1L-8) in the cells where these effects are stimulated in response to intracellular signal transmission after self-P55-1C.

The fact that the P55-1C capable of inducing ligand (TNF) - independent stimulation of other intracellular effects, such as inducing the expression of the gene 1L-8, means that the P55-1C or specific fragments can be used as a highly specific tool for the implementation of the desired effects in cells or tissues, without, however, treatment of these cells or tissue TNF. In many pathological conditions (for example, malignant tumors) treatment of cells with tumor necrosis factor (TNF), especially in high doses, can lead to undesirable side effects, systemically induced TNF after its binding with its receptor. Thanks to the present invention, it was found that the P55-1C can mimic other specific TNF-induced effects (in addition to cytotoxicity), for example, the induction of 1L-8, which opens the possibility of kletka - or tissue-specific introduction of the P55-1C or specific fragments, which are capable of transmitting the signal to induce the desired specific intracellular effects, such as inducing 1L-8, and thereby makes it possible to avoid the buildings permanently, it is the emergence of side effects, often observed in the process TWF-processing.

(d) Ligand-independent stimulation citizeny effects in cells Nega induced intracellular domains and the "death domains" of the p55-TNF-R and PAS-R (FAS/APO1)

As for the cytotoxic activity of the intracellular domains of the P55 TNF-R and FAS-R (RS and FAS-1C), in accordance with the present invention it was also found that RS, its "domain of death" (55DD) and FAS-1C have the ability to ligand-independent stimulation Titorenko effect in HeLa cells. For this purpose, HeLa cells were transfected expressing vectors containing different designs or full-p55-TNF-R, its fragments, including RS and 55DD or from PAS-1C. In one of the experiments, HeLa cells were cotransfection constructs containing the P55 TNF-R (p55-R) and FAS-1C (in more detail, these structures and their receipt described above). The results of this study are presented in Fig.9 (a and b), where on the left side of panels a and b of figure 9 schematically shows the construction used for transfection of HeLa cells; the two middle columns of these panels (second and third from left) graphically presents the results of the expression of TNF receptor or FAS; and in the first part of the panels graphically presents the results of a study of the viability of the transfected cells. On figa presents the results for transfected to etoc HeLa, expressing (in the short-term expression) full-p55-R, P55-1C or its fragments, or, as a control, luciferase (LUC), and in all cases used the vector tetracycline-controlled expression. On FIGU presents results for the transfected HeLa cells expressing (in the short-term expression) only one FAS-1C or together with the P55-R; moreover, these results were obtained using the vector tetracycline-controlled expression. The chart depicted on figa and 9B, where there's no shading rectangles correspond to cells transfected into Prilutskii tetracycline (1 μg/ml)that inhibits the expression and the shaded rectangles correspond to cells transfected in the absence of tetracycline. The expression of TNF receptor was evaluated at 20 hours after transfection by ELISA using antibodies against the extracellular domain of the receptor (see left panels), and by determining the binding of radioactively labeled TNF cells (middle part of the panels). Cytocidal effect of transfected proteins was assessed 48 hours after transfection. Data are presented from experiments 1-3 with similar quantitative results, where each design was tested twice. "ND" means "not determined". From the results shown n figa and 9B, it is seen that if expression of only one RS was observed even higher cytotoxicity. A significant level of cytotoxicity was also observed when the expression of only the "domain of death" (55DD). In contrast, the expression of the fragments RS that were missing the "domain of death" or contained only a fragment, did not affect cell viability. Expression of FAS-1C did not lead to significant cytotoxicity, but it increased the cytotoxicity of co-expressed P55-R.

Example 3

Other proteins having the ability to bind to the intracellular domain of p55-TNF-R or FAS-R

Using the techniques described above in Example 1 were isolated and identified three protein has the ability to communicate with RS or FAS-1C.

Figure 10-12 schematically depicts a partial and preliminary nucleotide sequence of cDNA clones, designated P2, P9 and DD 11, respectively.

Clones F2 and F9 were isolated by screening libraries of mouse embryos using murine FAS-1C as "bait". Figure 10 schematically shows the partial nucleotide sequence of the F2-cDNA, which was sequenced. Figure 11 schematically shows the partial nucleotide sequence of 1724 grounds from F9-CDNA, which was sequenced. Analysis of binding properties of protein-coding is used by the clones F2 and F9 (P2 and F9 respectively), showed that:

a) F2 strongly interacts with RS and 55DD person with FAS-1C mice, but it weakly interacts with irrelevant control protein SNF1 and designed, as well as with relevant (i.e. considered in this invention) protein FAS-1C of the person;

b ) F9 much interaction with people. p 55-1C and murine FA-1C but weakly interacts with people. RA-1C (relevant protein) and irrelevant (i.e., not related to the present invention) SNF1 protein and is designed;

C) None of the F2 and F9 does not interact with people. RS, pGBT9 ("empty" vector "bait"), or people CD-40.

In addition, research databases for genes and proteins ("Gene Bank" and "Prcteinbank") showed that F2 and F9 are new proteins.

In this way, the F2 and F9 are new proteins, specific binding to FAS-1C and IRS.

Clone DD11 was isolated by screening of a HeLa library person to use as "bait" 55DD) Fig schematically shows a partial nucleotide sequence of 425 basis from DD11-cDNA, which was sequenced.

Clone DD11 has a length of approximately 800 nucleotides. Full transcript, whose length is approximately 12 KB, was probed using this clone. Analysis of binding properties of the protein encoded by clone DD11 showed that DD11 strongly interacts with 55DD (AMI is ocelote 326-414) (see Fig.9) and does not interact with deletion mutants in this domain, for example, "AC-404". DD11 also interacts with mouse and people. FAS-1C, and to some extent with designed. However, DD 11 does not interact with SNF1, nor with pGBT9 ("empty" vector"bait"). In addition, it was not detected in any of the data banks ("Gene Bank" and "Proteinbank"). thus DD 11 is a protein specifically binding to RS (551DD) and FAS-1C.

Example 4 Construction of a soluble dimeric receptors TNF

Based on the data obtained above in Example 2, on the basis of which it was established that the intracellular domain of the p55-R (P55-1C) and part ("domain of death"), and vnutricletocny domain of FAS/APO1 and part (also referred to as the "domain of death"), similar to the "domain of death" P55-1C, have the ability to self became evident that it is possible to construct a new TWF-receptors, which are capable of self (aggregation), and which are soluble. Such receptors can be hybrid proteins containing, basically, the entire extracellular domain of the p55-R, connected mainly with the complete intracellular domain or its "domain of death" p55-R or FAS/APO1. Such structures do not contain the transmembrane domain of the p55-R (FAS/AP01), and therefore they are soluble. In addition, due to their ability to self intracellular domains or their "domain of death"these hybrid designs will be capable of oligomerization with education, at least dimers (and possibly multimeric higher order) p55-R. accordingly, such dimeric TNF receptor (p55-R) will be able to contact at least two monomers natural homotrimer TNF, thereby to form a soluble TNF-receptor to bind with high avidity with its ligand (homotrimeric TNF).

Based on the above, can be designed four types of the P55 TNF-receptor hybrid proteins, each of which is capable of oligomerization and is soluble:

i) a Hybrid product formed between the extracellular domain of the P55, (ES) and the intracellular domain of the P55-R (P55-1C);

ii) a Hybrid product formed between ES and "domain of death" P55-1C (DD55);

iii) a Hybrid product formed between US and the intracellular domain of FAS/APO1 (1CFAS); and

iv) a Hybrid product formed between ES and "domain of death" 1CFAS (DDFAS).

In each of the above hybrid proteins ability to bind with TNF monomer is provided AS-part protein, whereas oligomerization (or, at least, dimerization) of each of these types of proteins is ensured by its "tail" part, which is any of the areas RS, DD55, 1CFAD or DDFAS.

To construct the above hybrid proteins can be used a standard technique recom is anantnag DNA which is now widely used by professionals (see for example, Sambrook and others, (1989) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). For these purposes may be used any suitable expressing vector (cloning or plasmid used for the expression of selected insertional DNA), which is derived from bacteria, bacteriophages, or viruses of animals, and which can be introduced in one or several stages, DNA encoding ES and one of the "tails", which is the P55-1C, DD 55, 1CFAS or DDPAS. Insertiona thus DNA encoding each of the hybrid protein can be placed under the control of different regulatory sequences of system cloning or plasmids, such as promoters, binding sites with the ribosome, binding sites of transcription factors, etc. the Choice of such regulating the expression of the sequences depends on the type expressing vector, and therefore the type of host cell (eukaryotic or prokaryotic), and which it is desirable to Express the hybrid proteins of the present invention. Preferred cells of the host (and thus expressing vectors) are eukaryotic cells, particularly mammalian cells.

A DNA molecule encoding each of the above hybrid of the s protein, can be obtained and insertion in expressing vector, the following procedures;

a) First, using standard techniques, design a series of oligonucleotides for use in polymerase chain reaction, where these oligonucleotides are;

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

2) ACGC GTC GAC TGT G,QT G.CC TGA QTC CTC (AS, antisense), SEQ ID NO: 2;

3) ACGC GTC GAC CGC TAG CAA CGG TGG AAG (1C55, sense), SEQ ID NO: 3;

4) TCA TCT GAG AAG ACT GGG (1C55, antisense), SEQ ID NO: 4;

5) ACGC GAG GAC AAG AGA AAG GAA HUNDRED CAG (1CFA, sense), SEQ ID NO: 5;

6) one HUNDRED GAC CAA GCT TTG GAT (1CFA, antisense), SEO ID NO: 6;

7) ACCC CTC CAC CCC CCC ACC CTC TAC CCC (DD55; sense), SEQ ID NO: 7;

8) ACGC GTC GAC GAT CTT CAC TTG ACT AAA (DDFA, sense), SEQ ID NO: 8.

b) Plasmid containing the clones full-P55-R and FAS/APO1 receptor, and obtained in our laboratory (see also simultaneously consider the application ER and Examples 1-3 of the present application), subject to the following manipulations with obtaining DNA fragments that encode each of the hybrid proteins, which then lignot in the above selected expressing vector;

i) To obtain a DNA fragment encoding IS, which is a component of all four hybrid proteins, implementing RAC on the plasmid carrying cDNA people P55, using the above oligonucleotides # 1 and # 2 (fragment size is 640 BP)./p>

ii) For hybrid product ES-S, carry out PCR on plyamide carrying cDNA people P55, using the oligonucleotide NF 3 and 4, resulting in getting a DNA fragment encoding S (size 677 BP), which is then mixed with ES, hydrolyzed enzyme Sall, and are ligated by a blunt ends in any expressing vector under the control of the appropriate promoter. The orientation of the sequence IS-S, insertions in the vector is checked by restriction hydrolysis and by sequencing.

iii) For hybrid product ES-1C FA produce 1CFAS using PCR on the plasmid carrying cDNA for FAS, using the oligonucleotide No. 5 and 6, resulting in the receive fragment size (448 BP), which is then cut by the enzyme Sall and mixed with IS cut by the enzyme Sall, then are ligated according to the blunt ends in expressing vector under the control of the appropriate promoter. Orientation insertional EC55-1CFAS in the vector is checked by restriction hydrolysis and sequencing.

iv) To obtain a hybrid product ES-DD55 produce a DNA fragment containing DD 55-sequence using PCR on cDNA coding cell, and using the oligonucleotide No. 7 and 4. The resulting product size 314 BP cut by the enzyme Sall and mixed with AS cut DD11, then are ligated according to upim ends in expressing vector mammal. Orientation insertional product ES-DD55 in the vector is checked by restriction hydrolysis and sequencing.

v) To obtain a hybrid product ES-DD FAS produce a DNA fragment encoding DDFAS using PCR for FAS-cDNA using the oligonucleotide No. 6 and 8. The resulting product size 332 BP cut by the enzyme Sall and mixed with ES, hydrolyzed all, then are ligated according to the blunt ends in expressing vector mammal. Orientation insertional product EC55-DDFAS in the vector is checked by restriction hydrolysis and sequencing.

After constructing the above expressing vectors they can be entered in order of their expression, in a suitable mammalian cells (e.g. cells of the Chinese hamster ovary (Cho), or cells, monkey kidney (COS)) using standard methods. Expressed thus hybrid proteins can be purified by standard methods (see simultaneously applying AIR; EP 398327; and ER). Purified hybrid protein can then be analyzed for their ability to oligomerization (and the degree of oligomerization, i.e. the ability of proteins to form dimers or multimer higher order), as well as their ability to bind to TNF (and the affinity or avidity such binding).

Example 5

Design is Rurouni soluble dimeric receptors, FAS/APO1

In a manner analogous to the method described in Example 4, can be produced four types of hybrid FAS/ARE-proteins, each of which is capable of oligomerization and is soluble; namely:

i) a Hybrid product formed between the extracellular domain of FAS/APO1 (EC FAS) and the intracellular domain of the P55-1C;

ii) a Hybrid product formed between the EU FAS and the "domain of death" P55-1C (DD 55);

iii) a Hybrid product formed between the EU FAS and the intracellular domain of FAS/APO1 (1C FAS); and

iv) a Hybrid product formed between the EU FAS and the "domain of death" 1C FAS (FAS DD).

In each of the above hybrid proteins ability to bind to FAS ligand is provided by the EU FAS-part protein, whereas oligomerization (or, at least, dimerization) of each of these types of hybrid proteins by its "tail" part, which is any of the areas P55-1C, DD55, 1C, FAS, or FAS DD.

Design DNA fragments coding for the above-mentioned hybrid protein, and expressing the vectors containing these constructs, can be obtained as described in Example 4, except that this should be used other suitable oligonucleotides (not shown), after which these constructs can be used to obtain a fragment of the EU FAS intended for ligating with any of the above "tails is x" parts. After expressing vectors can be introduced into a suitable cell host, and the resulting expression of the hybrid proteins can be purified and analyzed for their ability to oligomerization (and the degree of oligomerization, i.e. on the ability of these proteins to form dimers or multimer higher order), as well as their ability to bind to FAS-ligand (and the affinity or avidity such binding).

Example 6

Construction of soluble oligomeric "mixed" receptors TNF/FAS

In order to obtain oligomeric receptors, with "mixed" affinity, i.e. the affinity of the two ligands, TNF and FAS-R ligand, the above 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 the TNF-R P75 TNF or the P55-TNF-R), which is connected with one of the P55-1C, FAS-1C, p55DD, or FASDD;

ii) obtaining a hybrid product, described in Example 5 and containing the extracellular domain of FAS-R, which is connected with one of: RS, FAS-1C, 55DD, or PAS-DD; and

iii) mixing any one of the hybrid products (i) with any one of the hybrid products (ii) to obtain a new dimeric (or oligomeric higher order) receptor, which contains both the extracellular domain of TNF-R and FAS-R, United through them-1C or-DD-regions.

In the above procedures, hybrid products (i) and (ii) can be obtained separately, namely, as a result of their selection of transformed cells, in which they were produced, and then these products can be mixed in vitro with the formation of the receptor with "mixed" affinity. Alternative cell-hosts can be transfected with vectors carrying sequences encoding hybrid proteins of both types; and in this case the receptor with "mixed" affinity can be obtained directly from the co-transfected cells. In fact, the oligomerization of hybrid products with the formation of oligomeric receptors can occur in cells during or after the procedure, the result can be obtained hybrid products, expressed in cells. For a specific selection of receptors with "mixed" affinity can be applied by any known method, for example, affinity chromatography in which to identify receptors with extracellular domains of both types are used in sequential chromatographic stages, antibodies against the extracellular domains of TNF-R and FAS-R.

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1. The selected DNA molecule encoding a polypeptide capable of contact with the intracellular domain of the p-55 TNF-receptor, which is characterized by a nucleotide sequence that encodes a polypeptide with the amino acid sequence corresponding to amino acid residues 328-426 SEQ ID No. 25, or a polypeptide with the amino acid posledovatel the activities the corresponding amino acid residues 277-426 SEQ ID No. 25.

2. The selected DNA molecule according to claim 1, characterized in that the nucleotide sequence is a cDNA available from cDNA libraries of human rights.

3. The selected DNA molecule according to claim 1 or 2, characterized in that it encodes a polypeptide with the amino acid sequence corresponding to residues 328-426 SEQ ID No. 25, designated as 55.1.

4. The selected DNA molecule according to claim 1 or 2, characterized in that it encodes a polypeptide with the amino acid sequence corresponding to residues 277-426 SEQ ID No. 25, designated as 55.3.

5. The expression vector containing a regulatory sequence operatively associated with a DNA molecule according to any one of claims 1 to 4.

6. The method of producing the polypeptide has the ability to communicate with the intracellular domain of the p-55 TNF-receptor involving culturing host cells transformed by the expression vector according to claim 5, under conditions suitable for expression of the specified polypeptide, and the selection of the target product.

7. The polypeptide having the ability to bind to the intracellular domain of the p-55 TNF-receptor and characterized by the amino acid sequence encoded by the DNA molecule according to any one of claims 1 to 4.

8. The polypeptide according to claim 7, characterized in that it has the amino acid sequence, according to stuudy balances 328-426 SEQ ID No. 25 (55.1).

9. The polypeptide according to claim 7, characterized in that it has an amino acid sequence corresponding to residues 277-426 SEQ ID No. 25 (55.3).



 

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SUBSTANCE: invention relates to a method based on phage display for preparing peptides interacting specifically with mammary Ehrlich tumor and can be used in therapy and diagnosis of malignant neoplasm. Peptides are prepared by affinity selection from phage peptide libraries comprising ten millions of different peptides of size 15 amino acid residues, the group of nine peptides wherein each peptide shows ability for accumulation in Ehrlich tumor. For practice using mimetic-peptides selected by such manner can be prepared by chemical synthesis and to use for preparing conjugates on their basis with the known cytotoxic preparations, radioactive isotopes and they can be incorporated in the composition of liposomal preparations for visualization of tumor neoplasm also.

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23 cl, 67 dwg, 1 tbl, 35 ex

FIELD: medicine, genetic engineering.

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FIELD: biotechnology, genetic engineering, immunology.

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27 cl, 13 dwg, 5 tbl, 8 ex

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EFFECT: improved preparing method, valuable properties of polypeptide.

10 cl, 4 dwg, 21 tbl, 12 ex

FIELD: biotechnology, genetic engineering, pharmaceutical industry.

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52 cl, 14 dwg, 3 tbl

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EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

FIELD: genetic engineering, molecular biology.

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EFFECT: improved isolating method, valuable biological properties of protein.

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FIELD: biochemistry, medicine.

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EFFECT: valuable medicinal properties of peptides.

1 tbl, 3 dwg, 4 ex

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