Patent EA-004309-B1 (EA)
Title: Modulators of thf receptor associated factor (traf), their preparation and use
Inventor: WALLACH DAVID
IPC: C12N15/113
Filed: 1997-04-01
Abstract: 1. A polypeptide that binds to TRAF2 and modulates the activity of NF-KB, said polypeptide comprising a) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO:7(NIK); b) an amino acid sequence of a fragment of a), which fragment binds to TRAF2 and modulates the activity of NF-KB, c) an amino acid sequence of an analog of a) or b), having no more than ten changes in the amino acid sequence of a) orb), each said change being a substitution, deletion or insertion of an amino acid, which analog binds to TRAF2 and modulates the activity of NF-KB; or d) a derivative of a), b) or c) which binds to TRAF2 and modulates the activity of NF-KB. 2. A polypeptide in accordance with claim 1, wherein said polypeptide of is the sequence encoded by the nucleotide sequence of SEQ ID NO:3.(clone 10) 3. A polypeptide in accordance with claim 1, wherein said polypeptide of is NIK (SEQ ID NO:7). 4. An NIK polypeptide according to claim 3, wherein said polypeptide has at least part of the amino acid sequence of SEQ ID NO: 7. 5. A DNA sequence encoding a polypeptide that binds to TRAF2 and modulates activity of NF-KB, selected from the group consisting of (1) a cDNA sequence comprising the nucleotide sequence of SEQ ID NO:3; (2) a cDNA sequence comprising the nucleotide sequence of SEQ ID NO:3; (3) a DNA sequence capable of hybridization to a sequence of (1)-(2) under moderately stringent conditions and which encodes a polypeptide that binds to TRAF2 and modulates the activity of NF-KB. 6. A DNA sequence in accordance with claim 5, comprising the nucleotide sequence of SEQ ID NO:3. 7. A DNA sequence in accordance with claim 5, comprising a DNA sequence encoding the protein NIK of SEQ ID NO:7. 8. A DNA sequence capable of hybridization to a sequence under moderately stringent conditions and which encodes a polypeptide. 9. A vector comprising a DNA sequence according to any one of claims 5. 10. A vector according to claim 9 capable of being expressed in a eukaryotic host cell. 11. A vector according to claim 9 capable of being expressed in a prokaryotic host cell. 12. Transformed eukaryotic or prokaryotic host cells containing a vector according to claim 9. 13. A method for producing a TRAF-binding protein, isoform, fragment, analog or derivative thereof, which comprises growing a transformed host cell according to claim 12 under conditions suitable for the expression of said protein, isoform, fragment, analog or derivative thereof, effecting post-translational modification, as necessary, for obtaining said protein, isoform, fragment, analog or derivative thereof, isolating said expressed protein isoform, fragment, analog or derivative. 14. Antibodies or active fragment or derivatives thereof, specific for a polypeptide according to claim 1. 15. A method for the modulation or mediation in cells of the activity of NF-KB or any other intracellular signaling activity modulated or mediated by TRAF2, said method comprising treating said cells by introducing into said cells one or more of said polypeptide in accordance with claim 1 in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more polypeptide 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. 16. A method according to claim 15 wherein said treating of said cells is by transfection of said cells with a recombinant animal virus vector comprising the steps of: (a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein (ligand) that is capable of binding to a specific cell surface receptor on the surface of said cells to be treated and a second sequence encoding a protein selected from said proteins, isoforms, analogs, fragments, and derivatives, that when expressed in said cells is capable of modulating/mediating the activity of NF-KB or any other intracellular signaling activity modulated/mediated by TRAF2 or other said molecules; and (b) infecting said cells with said vector of (a). 17. A method for modulating the TRAF2 modulated/mediated 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 a polypeptide according to claim 1, 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 TRAF2-binding protein in said cells. 18. for isolating and identifying a polypeptide according to any one of claims 1 to 4, capable of binding directly to TRAF2, comprising applying a yeast two-hybrid procedure in which a sequence encoding said TRAF2 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 TRAF2. 19. A method according to claim 15, wherein said protein is HIK or at least one of one isoform, fragment, analog or NIK derivative. 20. A pharmaceutical composition for the modulation of TRAF2 modulated/mediated effect on cells comprising, as active ingredient at least one polypeptide according to claim 1. 21. A pharmaceutical composition for modulating TRAF2 modulated/mediated effect on cells comprising, as active ingredient, a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding at least one polypeptide according to claim 2. 22. A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-KB induction or with any other activity mediated by TRAF2 or by other molecules to which a protein encoded by clone 10 according to claim 1 binds, said composition contains an effective amount of a protein encoded by clone 10. (SEQ ID NO:3). 23. A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-KB induction or with any other activity mediated by TRAF2 or by other molecules to which a protein encoded by clone 10 according to claim 1 binds, said composition contains an effective amount of DNA molecule, encoding protein, encoded by clone 10 (SEQ ID NO:3). 24. A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-KB induction or with any other activity mediated by TRAF2 or by other molecules to which a polypeptide according to claim 1 binds, said composition comprising an effective amount of a NIK protein, isoform, fragment, analog or derivative thereof. 25. A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-KB induction or with any other activity mediated by TRAF2 or by other molecules to which a polypeptide according to claim 1 binds, said composition comprising an effective amount of a DNA molecule, encoding NIK protein, isoform, fragment, analog or derivative thereof. 26. A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-KB induction or with any other activity mediated by TRAF2 or by other molecules to which a NIK protein, isoform, fragment, analog or derivative thereof according to claims 3 binds, said composition comprising an effective amount of a NIK protein, isoform, fragment, analog or derivative thereof. 27. A pharmaceutical composition for the prevention or treatment of a pathological condition associated with NF-KB induction or with any other activity mediated by TRAF2 or by other molecules to which a NIK protein, isoform, fragment, analog or derivative thereof according to claims 3 binds, said composition comprising an effective amount of a DNA molecule encoding NIK protein, isoform, fragment, analog or derivative thereof. 28. A method for the prevention or treatment of a pathological condition associated with NF-KB induction or with any other activity mediated by TRAF2 or by other molecules to which a polypeptide according to claim 1 binds, said method comprising administering to a patient in need an effective amount of polypeptide according to claim 1. 29. A method for the prevention or treatment of a pathological condition associated with NF-KB induction or with any other activity mediated by TRAF2 or by other molecules to which a polypeptide according to claim 1 binds, said method comprising administering to a patient in need an effective amount of a DNA molecule encoding polypeptide according to claim 1. 30. A method according to claim 28 or 29 wherein said protein is encoded by clone 10. 31. A method according to claim 28 or 29 wherein said protein is NIK. 32. A method for screening of a ligand capable of binding to a polypeptide according to claim 1 comprising contacting an affinity chromatography matrix to which said polypeptide is attached with a cell extract whereby the ligand is bound to said matrix, and eluting, isolating and analyzing said ligand. 33. A method for screening of a DNA sequence coding for a ligand capable of binding to a polypeptide according to claim 1 comprising applying the yeast two-hybrid procedure in which a sequence encoding said polypeptide is carried by one hybrid vector and sequences from a cDNA or genomic DNA library are carried by the second hybrid vector, transforming yeast host cells with said vectors, isolating the positively transformed cells, and extracting said second hybrid vector to obtain a sequence encoding said ligand. 34. A method for identifying and producing a ligand capable of modulating the cellular activity modulated