KR970703419A - Tumor Necrosis Factor / Nerve Growth Factor Superfamily Receptor and Water Soluble Oligomeric Modulators of Tumor Necrosis Factor / Nerve Growth Factor Superfamily Receptor - Google Patents

Abstract

The present invention generally relates to novel proteins that bind to the intracellular domains of p55 and p75 TNF-Rs and FAS-R, which modulate the functions of p55 and p75 TNF-Rs and FAS-R and the DNA sequences encoding them. You can. The present invention relates to oligomeric receptors having novel water soluble oligomeric TNF-Rs, oligomeric FAS-Rs, and mixtures of TNF-Rs and FAS-R. Furthermore, the present invention relates to the preparation and use of all of the above.

Description

Tumor Necrosis Factor / Nerve Growth Factor Superfamily Receptor and Water Soluble Oligomeric Modulators of Tumor Necrosis Factor / Nerve Growth Factor Superfamily Receptor

[Brief Description of Drawings]

1a to 1c diagrammatically depict partial and preliminary nucleotide sequences of cDNA clones encoding p55IC and p75IC binding proteins. Wherein Figure 1 (a) is the sequence of clone 55.11 encoding p55IC-binding protein 55.11; 1 (b) is a partial and preliminary sequence of clone 75.3 encoding p75IC-binding protein 75.3; 1 (c) is a partially preliminary sequence of clone 75.16 encoding p75IC-binding protein p75.16; The above is as described in Example 1; Figure 1 (d) depicts the putative amino acid sequence of protein 55.11 as estimated from the nucleotide sequence of Figure 1 (a) and is as described in Example 1.

FIG. 2 is a Northern Blot Reproduction showing 55.11-specific mRNAs seen in several tested cell lines, as described in Example 1. FIG.

3a and 3b are reduction of autoradiograms showing in vitro binding of a 55.11 cDNA encoded protein to a GST fusion protein comprising a portion of p55-IC. Here, in Figure 3a, the binding of the full-lengt 55.11 protein with various GST fusion proteins is depicted; And FIG. 3b depicts the binding of various GST fusion proteins with a portion of 55.11 fused to FLAG octapeptide, all as described in Example 1.

Figure 4 graphically shows a comparison of the putative amino acid sequence of human 55.11 with the relevant protein sequence of low evolutionary organisms. This is as described in Example 1.

FIG. 7 depicts the initiation of a ligand-independent cytotoxic effect in HeLa cells transfected with a full length p55-R, its intracellular domain, or part of an intracellular domain comprising the following death domain.

Since this is an open matter, no full text was included.

Claims (78)

A DNA sequence encoding a protein capable of binding to one or more intracellular domains of one or more receptors belonging to a tumor necrosis factor / nerve growth factor receptor superfamily. The DNA sequence of claim 1, wherein said receptor is TNF-Rs, p55, TNF-Rs or p75 TNF-Rs, or Fas-ligand receptor (FAS-R). The DNA sequence of claim 1 or 2, wherein the DNA sequence is selected from the group consisting of: (a) cDNA sequence derived from the coding portion of an intact TNF-R or FAS-R intracellular domain-binding protein: (b) normal DNA sequences that can hybridize with the sequences of (a) under stringent conditions and whose biological activity encodes a TNF-R or FAS-R intracellular domain-binding protein; And (c) a DNA sequence encoding a TNF-R or FAS-R intracellular domain binding protein that is biologically active and degenerate with the results of the genetic code of the DNA sequence defined in (a) and (b). . The DNA sequence of claim 1, wherein the DNA sequence comprises a p55 TNF-R cine domain (p55IC) -binding protein. DNA sequence encoding a protein selected from the group comprising proteins 55.1, 55.3, 55.11, F2, F9 and DD11 as named in this document. The DNA sequence of claim 5 selected from the sequences contained in cDNA clones 55.1, 55.3, 55.11, F2, F9, and DD11 as named herein. The DNA sequence of claim 1, which encodes a p75 TNF-R intracellular domain binding protein. 8. The DNA sequence of claim 7 encoding a protein selected from the group comprising proteins 75.3 and 75.16 as named herein. The DNA sequence of claim 8 selected from the sequences contained in cDNA clones 75.3 and 75.16 as named herein. 7. The DNA sequence of claim 5 or 6, wherein the protein having an amino acid sequence of residue 426 at amino acid residue 328 in the p55 TNF-R amino acid sequence encodes 55.3. 7. The DNA sequence of claim 5 or 6, encoding protein 55.3 having an amino acid sequence of residue 426 at amino acid residue 277 in the p55 TNF-R amino acid sequence. 7. The DNA sequence of claim 5 or 6, encoding protein 55.11 comprising the sequence depicted in Figure 1 (a). 10. The DNA sequence of claim 8 or 9, encoding protein 75.3 comprising the sequence depicted in FIG. 1 (b). 10. The DNA sequence of claim 8 or 9, encoding protein 75.16 comprising the sequence depicted in FIG. 1 (c). The DNA sequence of claim 1, which encodes a FAS-R intracellular domain (Fas-IC) binding protein. The DNA sequence of claim 15 encoding a protein selected from the group comprising proteins F2, F9 and DD11 as named herein. The DNA sequence of claim 16 selected from the sequences included in cDNA clones F2, F9, and DD11 as named herein. 18. The DNA sequence of claim 16 or 17, wherein the DNA sequence comprises a sequence selected from any one of FIGS. 10-12, and encodes any one of proteins F2, F9, and D11. 19. A sequence according to any one of the preceding claims, wherein the sequence according to one of the above proteins, analogs and derivatives capable of binding one or more intracellular domains of one or more TNF-Rs or FAS-R. Encoded proteins or analogs and derivatives. The protein of claim 19 selected from the group comprising proteins 55.1, 55.3, 55.11, 75.3, 75.16, F2, F9 and DD11, and analogs and derivatives thereof biologically active. The protein of claim 20, having the putative amino acid sequence depicted in FIG. 1 (d). 22. The protein of claim 21 characterized by binding to the intracellular domain of p55 TNF-R (p55IC) in the region between residues 243-308 of p55-TNF-R. A vector comprising a DNA sequence according to any one of claims 1 to 18. The vector of claim 23, which can be expressed in eukaryotic host cells. The vector of claim 23, which can be expressed in prokaryotic host cells. A transformed eukaryotic or prokaryotic host cell comprising a vector according to any one of claims 23 to 25. Growing the host cell transformed according to claim 26 under conditions suitable for expression of said protein, analogue or derivative, performing post-translational modification of said protein necessary for obtaining said protein and subjecting said published protein, analogue or derivative to said 21. A method for producing a protein analogue or derivative according to claim 19 or 20, comprising extracting from a culture medium of transformed cells or from a cell extract of said transformed cells. An antibody or active fragment or derivative thereof specific for a protein analogue or derivative according to claim 19 or 20. Proteins, analogs and derivatives according to claims 19 or 20 and analogs or derivatives thereof of protein p55IC, p55DD, FAS-IC or FAS-DD All of these proteins bind to the intracellular domain of the TNF-R or FAS-R Treatment of the cell with one or more proteins, analogs or derivatives selected from the group consisting of these proteins, wherein the treatment of the cell is in a form suitable for its introduction into the cell. Or introducing more proteins, analogs or derivatives into said cell or introducing a DNA sequence encoding said one or more protein analogs or derivatives in the form of a vector suitable for carrying said sequence, said vector being said sequence Depending on how it is expressed in the cell, the sequence can be effectively inserted into the cell. A method of modulating the effect of TNF or FAS-R ligands on TNF-R or FAS-R carrier cells, including methods that can be used. 30. The method of claim 29, wherein said treating said cells comprises the following steps: (a) specific for a TNF-R or FAS-R-carrying cell surface A sequence encoding a viral surface protein (ligand) capable of binding to a cell surface receptor and FAS-DD, an analog or derivative thereof according to claim 19 or 20 and P55IC, P55DD, FAS-IC or FAS-DD, its Analogs or derivatives, the protein may modulate the activity of the TNF-Rs or FAS-R when expressed in the cell, comprising: constructing a recombinant animal viral vector carrying a second sequence encoding a protein selected from the protein; And (b) infection of the cells with the vector of (a). A method of modulating TNF or FAS-R ligand effects in cells carrying one TNF-R or one FAS-R, comprising treatment of said cells with an antibody according to claim 28 or an active fragment or derivative thereof, The treatment is to apply an appropriate structure comprising the antibody, active fragment or derivative thereof of the cell, and when the IC binding protein is exposed to the surface of the cell, the structure is formed for application of the cell, and When the IC binding protein is intracellularly, the casting is formed for intracellular application. An oligonucleotide selected from a sequence according to any one of claims 1 to 18 and a sequence encoding an antisense sequence of p55IC, p55DD, FAS-IC or FAS-DD, wherein said oligonucleotide sequence comprises at least one TNF-R Or inhibit the expression of FAS-R intracellular domain binding protein, which modulates the effect of TNF or FAS-R ligands in TNF-R or FAS-R carrier cells, including treating the cells. Way. 33. The method of claim 32, wherein said oligonucleotide sequence is introduced into said cell via the virus of claim 30, wherein said second sequence of said virus encodes said oligonucleotide. A method of treating cancer cells or HIV-infected or other diseased cells, comprising: (a) a viral surface protein capable of binding to a specific cancer cell surface receptor or a receptor carried by an HIV-infected cell surface receptor or other diseased cell; The coding sequence and the proteins, analogs or derivatives of claims 19 and 20 and the p55TNF-R intracellular domain (p55IC, its "killing domain" (p55DD), the intracellular domain of FAS-R (FAS-IC) or " A death domain "(FAS-DD) or a biologically active analog or derivative of said protein, which is capable of killing said cell when expressed in said cancer, HIV infection or other diseased cell, the sequence encoding the protein selected therefrom (B) infecting the cancer or HIV-infected cells or other diseased cells with the vector (a). One or more proteins, analogs or derivatives thereof, essentially any self-binding intracellular domain protein capable of self binding of P55 TNF-R and inducing said TNF effect in cells in a ligand (TNF) -independent manner, or A method of inducing a TNF-related effect in a cell or tissue comprising treating the cell with a portion thereof, wherein the cell treatment comprises the one or more proteins, analogs or derivatives in a form suitable for introduction into the cell. Or a DNA sequence encoding said one or more proteins, analogs or derivatives into said cell in the form of a vector suitable for carrying said sequence, said vector in said cell depending on the manner in which said sequence is expressed in said cell. The sequence can be effectively inserted into. 36. The method of claim 35, further comprising the steps of: treating said cell by transfection of said cell with a recombinant animal virus vector comprising the following steps; (a) a recombinant animal carrying a sequence encoding a viral surface protein (ligand) capable of binding to a specific cell surface protein on the surface of the cell to be treated, and a second sequence encoding protein p55IC, a portion thereof, analogs and derivatives thereof Composition of the viral vector, the protein is self-binding when expressed in the cell and can induce the one or more TNF-related effects. (b) infection of said cells with the vector of (a). 37. The method of claim 35 or 36, wherein the TNF effect induced in the cell is the induction of IL-8 gene expression and the vector encodes essentially all of the p55-ICs, portions thereof, analogs and derivatives thereof. It carries a sequence and is a signal of induction of the IL-8 gene expression when expressed in self binding cells. 38. The method of any one of claims 35-37, wherein the method comprises the steps of treating cancer cells or virally infected cells or enhancing the antifungal effect of granulite, wherein the viral vector comprises the cancer cells, virally infected cells or granulocytes. Carries a sequence encoding a viral ligand capable of binding to a specific cell surface receptor on its surface and a sequence encoding the p55IC, a portion thereof, an analog or derivative thereof, and is expressed in the cancer, virus-infected or granulosite cells When induced, kills these cells, which induce TNF-related effects. The method of claim 38, wherein the p55IC, a portion thereof, analogue or derivative thereof induces expression of IL-8 when expressed in cancer cells and kills the cancer cells by their chemotactic activity Granulocytes and other lymphocytes are attracted to cancer cells, causing cancer cells to die. P55-R intracellular domain (p55IC), parts thereof, analogs and derivatives thereof for inducing TNF-related effects and for use in cell therapy. The p-55IC, part, analogues and derivatives of claim 40 for use in treating cells by inducing IL-8 gene expression. 42. The p55IC, daily report, analogues and derivatives for use in treating cancer cells by inducing expression of the FL-8 gene resulting in the death of cancer cells. The vector encoding the ribozyme sequence encoding a ribozyme sequence capable of interacting with a cellular mRNA encoding a protein according to claim 19 or 20 or an mRNA encoding p55IC, p55DD, FAS-IC or FAS-DD. Introduced into the cell in a form that can be expressed in the cell, the ribozyme sequence is expressed in the cell and interacts with the cell mRNA sequence, and the protein or the p55IC, p55DD, FAS-IC or FAS- A method of modulating a TNF or FAS-R ligand effect in a cell comprising the application of a ribozyme method for cleaving said mRNA sequence that inhibits intracellular expression of DD. 21. A method for isolating and identifying a protein, element or receptor capable of binding to an intracellular domain binding protein according to claim 19 or 20, wherein the protein according to claim 19 or 20 is attached to an adsorption chromatography substrate, The attached protein is contacted with the cell extract, and the protein, urea or receptor of the cell extract bound to the attached protein comprises a method of application of adsorption chromatography, which is then eluted, separated and analyzed. Sequences encoding intracellular domain binding proteins are carried by one hybrid vector, sequences from cDNA or genomic DNA libraries are carried by a second hybrid vector, and the vectors are then used to transform yeast host cells and are positive. The cell according to claim 19 or 20, wherein the transformed cell is isolated and applying a yeast double hybridization method by extracting said second hybrid vector to obtain a sequence encoding a protein that binds said intracellular domain binding protein. How to isolate and identify proteins that can bind to my domain binding proteins. 21. TNF- or FAS of a cell, comprising the other protein or protein p55IC, p55DD, FAS-IC or FAS-DD thereof as its activator, according to claim 19 or 20. Pharmaceutical Compounds for Modulation of -R Ligand Effect. A recombinant animal viral vector encoding a protein capable of binding to a cell surface receptor and encoding a protein according to claim 19 or 20, or a protein p55IC, p55DD, FAS-IC or FAS-DD, a biologically active fragment or analog thereof A pharmaceutical compound for modulating the TNF- or FAS-R ligand effect of cells composed of activators. A pharmaceutical compound for modulation of the TNF- or FAS-R ligand effect in a cell composed of an oligonucleotide encoding an antisense sequence of the sequence according to any one of claims 1 to 18 as an activator. 19. A method of isolating and identifying proteins capable of binding to TNF-Rs or FAS-R intracellular domains comprising applying PCR cloning methods following non-stringent Southern hybridization, wherein any one of claims 1-18 The sequence according to claim 1 or a portion thereof is used as a probe for binding with a sequence of a cDNA or genomic DNA library, has a minimum partial similarity to it, and the binding sequence is then amplified and cloned by PCR method. A clone encoding the sequence having minimal partial similarity to said sequence of claim 18 is obtained. Water-soluble oligomeric tumor necrosis factor receptor (TNF-R) and salts and functional derivatives thereof comprising at least two self-binding fusion proteins, each fusion protein comprising (a) an extracellular domain, analog or derivative of TNF-R at one end Has a TNF binding domain, wherein the domain, analog or derivative of the cell is incapable of detrimental self-binding and thus inhibits TNF binding or results in lower binding than TNF optimal binding and can bind to TNF; (b) at the other end (i) essentially the intracellular domain of mode p55TNF-R (p55IC), which extends from about 206 to about 426 amino acid residues of an intact p55TNF-R molecule (p55-R); (ii) the killing domain of p55-IC extending amino acid residues from about 328 to 426 intact p55-R; (iii) the intracellular domain of essentially all Fas / APO1 receptors (FAS-IC); (iv) the death domain of FAS-IC; And (v) analogs, aliquots, or derivatives of any of (i) to (iv) having a self-binding ability; A water-soluble oligomeric TNF receptor having a self-binding domain selected from wherein the at least two self-binding proteins self-bond only at the end (b) and have the end (a) capable of binding at least two TNF monomers, Each terminal (a) may bind with one TNF monomer. 51. The composition of claim 50, wherein at least two termini (a) and essentially all of said extracellular domains of essentially all p55 TNF-Rs (p55-R) that extend from amino acid residues 1 to about 172 of intact p55-R A water soluble oligomer TNF-R comprising at least two termini (b) that is p55-IC. 51. The method of claim 50, wherein at least two termini (a) which are essentially extracellular domains of all p55-Rs extending from amino acid residues 1 to about 172 of intact p55-R and essentially all of the kill domains of p55-IC A water soluble oligomer TNF-R comprising at least two termini (b). 51. The composition of claim 50, wherein the analogs or derivatives of the domains of essentially all of the p55-R's cells that are capable of binding TNF monomers and are unable to self-bond, at least two termini (a) which are analogs or derivatives to each of the above A water-soluble oligomer TNF-R comprising at least two termini (b) that are p55-IC. 51. The composition of claim 50, wherein the analogs or derivatives of the domains of essentially all of the p55-R's cells that are capable of binding TNF monomers and are unable to self-bond, at least two termini (a) which are analogs or derivatives to each of the above A water soluble oligomer TNF-R comprising at least two termini (b) of the death domain of p55-IC. 51. The method of claim 50, wherein at least two termini (a) are essentially the extracellular domains of essentially all p55-Rs extending from amino acid residues 1 to about 172 of intact p55-R and at least two are essentially all of the above FAS-IC. Water soluble oligomer TNF-R comprising two ends (b). 51. The composition of claim 50, wherein at least two termini (a) which are essentially the extracellular domain of essentially all p55 TNF-Rs (p55R) extending from amino acid residues 1 to about 172 of intact p55-R and essentially all of the FAS- A water soluble oligomer TNF-R comprising at least two termini (b), the killing domain of the IC. 51. The composition of claim 50, wherein at least two termini (a) and essentially all analogs or derivatives of essentially all of the extracellular domains of p55-R that are capable of binding TNF monomers and are not self-bonding A water-soluble oligomer TNF-R comprising at least two termini (b) which are said FAS-IC. 51. The composition of claim 50, wherein at least two termini (a) and essentially all analogs or derivatives of essentially all of the extracellular domains of p55-R that are capable of binding TNF monomers and are not self-bonding A water-soluble oligomer TNF-R comprising at least two termini (b), said killing domain being FAS-IC. 60. A method for producing a water soluble oligomer TNF-R according to any one of claims 50 to 58, comprising: (a) the construction of an expression vector encoding any one of the fusion proteins, each said end of the fusion protein The DNA sequence of is obtained from a cloned DNA sequence that essentially encodes the domains, analogs or derivatives of all TNF-R cells; And from essentially cloned DNA sequences encoding all of the p55IC, p55IC killing domains, Fas-IC or Fas-IC killing domains, analogs or derivatives thereof, the ends being linked together to form a fusion protein sequence The fusion protein sequence is inserted into the vector under the control of transcriptional and translational regulatory sequences. (b) introducing the vector of (a) into a suitable host cell in which said fusion protein is expressed; And (c) purifying the fusion protein expressed in the host cell, the fusion protein is self-bonded before, during, or after the purification process to obtain a water-soluble oligomer TNF-R. 60. An expression vector comprising a fusion protein sequence encoding said fusion protein of any one of claims 50-58. 61. A vector according to claim 60 for use in a method according to claim 59. A host cell containing a vector according to claim 60, wherein said fusion protein sequence can be expressed. 59. A pharmaceutical compound comprising a water soluble oligomer TNF-R, a salt or a functional derivative thereof, and a mixture thereof as an activator according to any one of claims 50 to 58 together with a pharmacologically acceptable carrier. 59. The method according to any one of claims 50 to 58, against the detrimental effects of TNF in mammals, in particular in the therapeutic conditions in which excess TNF is produced in vivo or in which excess TNF is administered externally. Water-soluble oligomer TNF-R, salts thereof or functional derivatives thereof and mixtures thereof. 59. The water soluble oligomer TNF-R, salt or functional derivative thereof according to any one of claims 50 to 58, which is used to sustain the beneficial effect of TNF in mammals when administering TNF ex vivo. mixture. A water-soluble oligomeric Fas / AP01 receptor (Fas-R) comprising at least two self-binding fusion proteins, each fusion protein comprising (a) at one end a Fas ligand binding domain selected from an extracellular domain, analog or derivative of Fas-R. They are incapable of self-bonding and can bind to the Fas ligand; (b) at the other end (i) essentially the intracellular domain of all p55 TNF-Rs (p55IC), which extend from about 206 to about 426 amino acid residues of an intact p55TNF-R molecule (p55-R); (ii) the killing domain of p55-IC with an amino acid residue of intact p55-R extending from about 328 to 426; (iii) essentially the intracellular domains of all Fas / APO1 receptors (Fas-IC); (iv) Fas-IC killing domain of Fas-IC; And (v) analogs, aliquots, or derivatives of any of (i) to (iv) having a self-binding ability; A water-soluble oligomer Fas / APO1 having a magnetic binding domain selected from among the receptors, wherein the at least two magnetic binding proteins are self-bonding only at the terminal (b), and the terminal (a) capable of binding at least two Fas monomers And each terminal (a) may bind to one Fas monomer, and the present invention includes salts and functional derivatives of the water-soluble oligomer Fas / APO1R. A process for producing a water soluble oligomer Fas-R according to claim 66, comprising: (a) the construction of an expression vector encoding any of the fusion proteins, the DNA sequence of each said end of the fusion protein is obtained from a DNA sequence which is essentially a clone encoding the extracellular domain, analog or analog of all Fas-Rs ; And from essentially cloned DNA sequences encoding all of the p55IC, p55IC killing domains, Fas-IC or Fas-IC killing domains, analogs or derivatives thereof, the ends being linked together to form a fusion protein sequence The fusion protein sequence is inserted into the vector under the control of the former and translational regulatory sequences. (b) The vector of (a) is inserted into the fusion protein sequence under the control of the translocation and translation region regulatory sequence. (b) introducing the vector of (a) into a suitable host cell in which said fusion protein is expressed; And (c) self-bonding before, during, or after purification of the fusion protein expressed in the host cell, during the purification of the fusion protein, to obtain a water-soluble oligomeric Fas-R. 67. The host cell of claim 66, wherein the fusion protein comprises a fusion protein sequence encoding. A vector according to claim 68 used in a method according to claim 67. A host cell comprising the vector of claim 68 capable of expressing said fusion protein sequence. A pharmaceutical composition comprising a water-soluble oligomeric Fas-R, a salt or a functional derivative thereof according to claim 66, and a pharmaceutically acceptable carrier thereof as an activator thereof. Water-soluble oligomer Fas-F, salts or functional derivatives thereof, for the purpose of countering the deleterious effects of Fas ligands in mammals according to claim 66 in therapeutic conditions in which Fas Ligard is over-in vivo or over-dosed externally; Mixtures thereof. 58. A water soluble oligomer receptor (mixed adsorption receptor) comprising at least two self-bonding fusion proteins, wherein the water-soluble oligomer receptor (mixed adsorption receptor) has adsorption to both TNF and FAS-R ligands, wherein one of the fusion proteins is TNF of any one of claims 50-58. The specific TNF-R-derived protein, and the other fusion protein is the FAS-R ligand-specific FAS-R-derived protein of claim 66. 74. A pharmaceutical composition comprising the mixed adsorption receptor according to claim 73. The mixed adsorption receptor according to claim 73 used for combating the deleterious effects of TNF and FAS-R ligands in mammals. A pharmaceutical compound according to claim 46 for treating a cell according to the induction of a TNF related effect, the pharmaceutical compound comprising p55IC as its activator, a portion thereof, an analog and a derivative, and a pharmacologically acceptable carrier. A pharmaceutical compound according to claim 57 for treating a cell according to the induction of a TNF related effect, the pharmaceutical compound comprising a recombinant animal virus and a pharmacologically acceptable carrier encoding p55IC, a portion thereof, analogs and derivatives as activators. 77. A pharmaceutical compound according to claim 76 or 77 for the purpose of treating cancer cells, the administration of said compound induces IL-8 expression resulting in cancer cell death. ※ Note: The disclosure is based on the initial application.