Patent EA-004658-B1 (EA)
Title: B1 protein capable to modulate intracellular inflammation, cell death and cell survival pathways, encoding dna molecule thereof, vector containing said dna molecule, cell strain containing said vector, methods for their use and pharmaceutical composition
Inventor: WALLACH DAVID
IPC: A61P43/00
Filed: 1998-06-01
Abstract: 1. A DNA molecule encoding a B1 with amino acid sequence depicted in Fig. 3, being capable of modulating with intracellular inflammation, cell death or cell survival pathways directly or indirectly, by association with other intracellular modulators or mediators of these pathways, and fragments and variants, fragments and derivatives of said DNA. 2. A DNA molecule according to claim 1, having a nuclear sequence depicted in Fig 3B. 3. An expressing vector according to claim 1 or 2, comprising a DNA molecule or a fragment thereof. 4. A vector according to claim 3 capable of being expressed in a eukaryotic host cell. 5. A vector according to claim 3 capable of being expressed in a prokaryotic host cell. 6. Strain of transformed eukaryotic host cells containing a vector according to any one of claims 3-5. 7. Strain of transformed prokaryotic host cells containing a vector according to any one of claims 3-5. 8. A B1 protein, being capable of modulating with intracellular inflammation, cell death or cell survival pathways directly or indirectly, by association with other intracellular modulators or mediators of these pathways, being encoded by a DNA molecule according to claim 1 or 2 and fragments and derivatives thereof obtained as said in the description. 9. The B1 protein according to claim 8, being encoded by a DNA molecule according to claim 2. 10. A method for producing a B 1 protein or fragments and derivatives thereof according to claim 6 or 9, which comprises growing a transformed host cell according to claim 6 or 7 under conditions suitable for the expression of said protein effecting post-translational modification and isolating said protein. 11. An antibody specific for the B1 protein, fragment or derivative thereof according to claim 8 or 9. 12. An antibody fragment according to claim 11. 13. A method for the modulation in cells of the activity of inflammation, cell death or cell survival pathways or any other intracellular signaling activity modulated or mediated by a B1 protein or a fragment thereof, or a derivative thereof according to claim 8 or 9 or by other molecules to which a B1 protein, or a fragment or a derivative binds or otherwise interacts, said method comprising introducing into said cells one or more of said B1 protein, fragment or derivative thereof in a form suitable for intracellular introduction thereof. 14. A method for the modulation in cells of the activity of inflammation, cell death or cell survival pathways or any other intracellular signaling activity modulated or mediated by a B1 protein or a fragment thereof, or a derivative thereof according to claim 8 or 9 or by other molecules to which a B1 protein, or a fragment or a derivative binds or otherwise interacts, said method comprising introducing into said cells vector according to any one of claims 3-5. 15. The method according to claim 14, characterized in that said vector is a recombinant animal virus vector, containing a nucleic acid molecule encoding a viral surface protein (ligand) that is capable of binding to a specific receptor on the surface of said cells to be treated. 16. A method for the modulation in cells of the activity of inflammation, cell death or cell survival pathways the activity of which is modulated or mediated by B 1 protein according to claim 8 or 9, and the B1 protein or fragments and derivatives thereof are exposed on the surface of said cells, said method comprising treating said cells with a pharmaceutical composition containing an antibody or a fragment thereof according to claim 11 or 12 prepared in the form suitable for an extracellular application. 17. A method for the modulation in cells of the activity of inflammation, cell death or cell survival pathways the activity of which is modulated or mediated by B 1 protein according to claim 8 or 9, having intracellular localization, said method comprising treating said cells with a pharmaceutical composition containing an antibody or a fragment thereof according to claim 11 or 12 prepared in the form suitable for an extracellular application. 18. A method for modulating the inflammation, cell death or cell survival or other pathways in which cells are modulated or mediated by a B1 protein, comprising introducing a recombinant animal virus vector into said cells, containing ribozyme sequence capable of interacting with a cellular mRNA sequence, encoding a B 1 protein according to claim 8 or 9, 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 B1 protein in said cells. 19. A pharmaceutical composition for the modulation of the inflammation, cell death, cell survival or other pathways in cells which are modulated directly or indirectly by B 1 protein according to claim 8 or 9, comprising, as active ingredient, a B1 protein or a fragment or a derivative thereof according to claim 8 or 9 or mixtures thereof. 20. A pharmaceutical composition for the modulation of inflammation, cell death, cell survival or other pathways in cells which are modulated directly or indirectly by B1, comprising, as active ingredient, a recombinant animal virus vector containing nucleic acid molecule, encoding a viral surface protein (ligand) that is capable of binding to a specific receptor on the surface of said cells to be treated. 21. A pharmaceutical composition for the prevention or treatment of a pathological condition associated with the regulation of apoptosis by one or more molecules to which a B1 protein, according to claim 8 or 9, binds directly or indirectly, said composition comprising a B1 protein or a fragment, or a derivative thereof as an effective ingredient according to claim 8 or 9. 22. A pharmaceutical composition is one for the prevention or treatment of a pathological condition associated with the regulation of apoptosis by one or more molecules to which the B1 protein, according to claim 8 or 9, binds directly or indirectly, said composition comprising a DNA molecule as an effective ingredient according to claim 1 or 2 or a fragment thereof or derivative thereof. 23. A method for screening of a ligand capable of binding to a B1 protein according to claim 8 or 9, comprising contacting an affinity chromatography matrix to which said protein is attached with a cell extract whereby the ligand is bound to said matrix, and eluting, isolating and analyzing said ligand. 24. A method for screening of a DNA sequence coding for a ligand capable of binding to a B1 protein according to claim 8 or 9, comprising applying the yeast two-hybrid procedure in which a sequence encoding said B1 protein 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. 25. A method for identifying and producing a ligand capable of modulating the cellular activity modulated/mediated by a B1 protein according to claim 8 or 9, comprising: a) screening for a ligand capable of binding to a polypeptide comprising at least a portion of a B1 protein having at least some of the amino acid residues of a B1 protein depicted in Fig. 3, which include essentially all of the prodomain (or CARD)of a B1 protein; b) identifying and characterizing a ligand, other than BCL2, TRAF2, or portions of a receptor of the TNF/NGF receptor family or other known proteins having a prodomain (CARD), found by said screening step to be capable of said binding; and c) producing said ligand in substantially isolated and purified form. 26. A method for identifying and producing a ligand capable of modulating the cellular activity modulated or mediated by a B1 protein according to claim 8 or 9 comprising: a) screening for a ligand capable of binding to a polypeptide comprising at least the carboxy terminal portion of the B1 protein sequence depicted in Fig. 3 including the prodomain (CARD); b) identifying and characterizing a ligand, other than BCL2, TRAF2, or portions of a receptor of the TNF/NGF receptor family or other known proteins having a prodomain (CARD), found by said screening step to be capable of said binding; and c) producing said ligand in substantially isolated and purified form. 27. A method for identifying and producing a ligand capable of modulating the cellular activity modulated/mediated by a B1 protein according to claim 8 or 9, comprising: a) screening for a ligand capable of binding to at least the N-terminal portion of the B1 protein sequence depicted in Fig. 3 including substantially all of the kinase domain of a B1 protein; b) identifying and characterizing a ligand, other than BCL2, TRAF2, or portions of a receptor of the TNF/NGF receptor family or other known intracellular modulatory proteins, found by said screening step to be capable of said binding; and c) producing said ligand in substantially isolated and purified form. A. Amino acid sequence in frame - kinase domain, underlined - CARD domain B. nucleotide sequence