Patent EA-003269-B1 (EA)
Title: Cash(caspase homologue) with death effector domain, modulators of the function of fas receptors
Inventor: WALLACH DAVID
IPC: A61P43/00
Filed: 1998-02-26
Abstract: 1. A DNA sequence encoding a polypeptide having the activity of a G1 protein, said polypeptide (i) being capable of binding to or interacting directly or indirectly with MORT-1 and/or any of the MORT-1-binding proteins, or other intracellular mediator/modulator proteins; and (ii) being capable of mediating the intracellular effect mediated by the FAS-R or p55-TNF-R, or other cytotoxic mediators or inducers; said DNA sequence selected from the group consisting of: (a) a DNA sequence encoding a G1 isoform having the amino acid sequence depicted in Fig. 1; (b) a DNA sequence encoding a G1 isoform having the amino acid sequence depicted in Fig. 2; (c) a DNA sequence encoding at least part of the polypeptide having the amino acid sequence as shown in Fig. 1 or Fig. 2; (d) a DNA sequence comprising at least part of the sequence depicted in Fig.1 and encoding at least one isoform of the G1 protein, the G1(, isoform; (e) a DNA sequence comprising at least part of the sequence depicted in Fig.2 and encoding at least one isoform of the G1 protein, the G1( isoform; (f) a cDNA sequence derived from the coding region of a native isoform of G1 protein; (g) a DNA sequence encoding a fragment, analog or derivative of a polypeptide encoded by the DNA sequence of any one of (a) to (f); (h) a DNA sequence capable of hybridization to a sequence of any one of (a) to (g) under moderately stringent conditions and which encodes a biologically active isoform of G1 protein; and (i) a DNA sequence which is degenerate as a result of the genetic code to the DNA sequences defined in any one of (a) to (h) and which encodes a biologically active isoform of G1 protein. 2. A vector comprising a DNA sequence according to claim 1. 3. The vector according to claim 2 capable of being expressed in a eukaryotic or prokaryotic host cell. 4. The vector according to claim 2 or 3 which is a recombinant animal virus vector carrying a DNA sequence encoding a viral surface protein (ligand) that is capable of binding a specific cell surface receptor on the surface of a FAS-R- or p55-R-carrying cell and a second sequence comprising the DNA sequence of claim 1. 5. The vector according to claim 4 wherein the cell surface receptor is specific to tumor cells, HIV-infected cells or other diseased cells. 6. A transformed eukaryotic host cell strain containing a vector according to any one of claims 2 to 5. 7. A transformed prokaryotic host cell strain containing a vector according to any one of claims 2 to 5. 8. A method for producing a polypeptide having the activity of a G1 protein comprising growing the transformed host cell strain according to claims 6 or 7 under conditions suitable for the expression of said polypeptide effecting post-translational modifications as necessary for obtaining of said polypeptide and isolating said polypeptide. 9. A polypeptide having the activity of a G1 protein, said polypeptide (i) being capable of binding to or interacting directly or indirectly with MORT-1 and/or any of the MORT-1-binding proteins, or other intracellular mediator/modulator proteins; and (ii) being capable of mediating the intracellular effect mediated by the FAS-R or p55-TNF-R, or other cytotoxic mediators or inducers; said polypeptide selected from the group consisting of: (a) a G1 isoform having the amino acid sequence depicted in Fig. 1; (b) a G1 isoform having the amino acid sequence depicted in Fig. 2; (c) at least part of the polypeptide having the amino acid sequence as shown in Fig. 1 or Fig. 2; (d) at least part of the sequence depicted in Fig.1 comprising at least one isoform of the G1 protein, the G1(, isoform; (e) at least part of the sequence depicted in Fig.2 and encoding at least one isoform of the G1 protein, the G1( isoform; and (f) a fragment, analog or derivative of a polypeptide according to any one of (a) to (e); or obtainable by the method of claim 8. 10. An antibody specific for the polypeptide according to claim 9. 11. An in vitro method for the modulation of cell death or inflammatory processes, comprising treating said cells with one or more polypeptides according to claim 9, wherein said treating of said cells comprises introducing into said cells said one or more polypeptides in a form suitable for intracellular introduction thereof, or introducing into said cells the DNA sequence of claim 1 in the form of suitable vector carrying said sequence, said vector capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells. 12. An in vitro method for the modulation of the FAS-R ligand or TNF effect on cells carrying a FAS-R or p55-R, comprising treating said cells with one or more polypeptides according to claim 9 wherein said treating of said cells comprises introducing into said cells said one or more polypeptides in a form suitable for intracellular introduction thereof, or introducing into said cells the DNA sequence of claim 1, encoding said one or more polypeptides in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells. 13. The method according to claim 12, wherein said treating of cells comprises introducing into said cells one of said polypeptides, said DNA sequence encoding one of said polypeptides in the form of a suitable vector carrying said sequence, said vector being capable of effecting the insertion of said sequence into said cells in a way that said sequence is expressed in said cells. 14. The method according to claim 12 or 13 wherein said treating of said cells is by transfection of said cells with the recombinant animal virus vector according to claim 4. 15. An in vitro method for the modulation of cell death or inflammatory processes, comprising treating said cells with one or more inhibitors of one or more proteins/enzymes mediating said cell death or inflammatory processes, said inhibitors being selected from the group consisting of: (i) one or more polypeptides according to claim 9 capable of inhibiting said cell death or inflammatory processes; and (ii) inhibitors of one or more polypeptides according to claim 9 when said one or more polypeptides augments/enhances or mediates said cell death or inflammatory processes. 16. An in vitro method for modulating the FAS-R ligand or TNF effect on cells carrying a FAS-R or a p55-R comprising treating said cells with the antibody according to claim 10, said treating being by application of a suitable composition containing said antibody to said cells, wherein when the G1 protein or portions thereof of said cells are exposed on the extracellular surface, said composition is formulated for extracellular application, and when said G1 proteins are intracellular said composition is formulated for intracellular application. 17. An in vitro method for modulating the FAS-R ligand or TNF effect on cells carrying a FAS-R or p55-R comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence for at least part of the DNA sequence according to claim 1, said oligonucleotide sequence being capable of blocking the expression of the G1 protein. 18. The method according to claim 17 wherein said oligonucleotide sequence is introduced to said cells via the vector of claim 4 wherein said second sequence of said virus encodes said oligonucleotide sequence. 19. A method for modulating the FAS-R ligand or TNF effect on cells comprising applying a ribozyme procedure in which a vector encoding a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding the polypeptide according to claim 9 is introduced into said cells in a form that permits expression of said ribozyme sequence in said cells, and wherein when said ribozyme sequence is expressed in said cells it interacts with said cellular mRNA sequence and cleaves said mRNA sequence resulting in the inhibition of expression of said polypeptide in said cells. 20. The method according to claims 11 or 15, wherein said polypeptide is capable of binding directly or indirectly to MORT-1 and/or to any MORT-1-binding protein, which MORT-1, in turn, binds specifically to FAS-IC, or which are capable of binding directly or indirectly to MORT-1 and/or any of the MORT-1-binding proteins, which, MORT-1, in turn, binds to TRADD, which, in turn, binds to the p55-IC. 21. The method according to any one of claims 12, 16 to 19, wherein said polypeptide is capable of binding directly or indirectly to MORT-1 and/or to any MORT-1-binding protein, which MORT-1, in turn, binds specifically to FAS-IC, or which are capable of binding directly or indirectly to MORT-1 and/or any of the MORT-1-binding proteins, which, MORT-1, in turn, binds to TRADD, which, in turn, binds to the p55-IC. 22. A method for isolating and identifying the polypeptide according to claim 9, comprising applying the yeast two-hybrid procedure in which a sequence encoding the MORT-1 protein and/or MORT-1-binding protein is carried by one hybrid vector and sequence from a cDNA or genomic DNA library is carried by the second hybrid vector, the vectors then being used to transform yeast host cells and the positive transformed cells being isolated, followed by extraction of the said second hybrid vector to obtain a sequence encoding a protein which binds to said MORT-1 protein, and/or MORT-1-binding proteins. 23. The method according to claims 11 or 15 wherein said polypeptide is at least one of the G1 isoforms, analogs, fragments and derivatives thereof. 24. The method according to any one of claims 12, 16 to 19 wherein said polypeptide is at least one of the G1 isoforms, analogs, fragments and derivatives thereof. 25. The method according to claim 22 wherein said polypeptide is at least one of the G1 isoforms, analogs, fragments and derivatives thereof. 26. An in vitro method for the modulation of the MORT-1-induced effect of MORT-1-binding protein-