Caspase-8 binding protein, its preparation and use

Abstract

The present invention relates to a caspase-8 interacting polypeptide (Cari), methods for its preparation, and its use.

1 . An isolated polypeptide capable of binding to pro-caspase-8 selected from the group consisting of:
 a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 3 (Cari);   b) a variant of Cari (SEQ ID NO: 3) comprising no more than ten amino acid changes and retains the capability of binding to pro-caspase-8:   c) a fragment of Cari (SEQ ID NO: 3) having the sequence of SEQ ID NO: 5 and includes the pro-caspase-8 binding site; and   d) a derivative of Cari (SEQ ID NO: 3), which is prepared by modifying the —NH 2  group at the N-terminus or the —COOH group at the C-terminus or the functional groups that appear as side chains of one or more amino acid residues of the polypeptides of (a) to (c), without changing one amino acid to another one of the twenty commonly occurring natural amino acids,   
       with the proviso that said polypeptide is not DF518-3. 
     
     
         2 . An isolated DNA sequence encoding a polypeptide in accordance with  claim 1 . 
     
     
         3 . A ribozyme specific for a DNA sequence according to  claim 2 . 
     
     
         4 . A DNA sequence encoding an antisense sequence to a DNA of  claim 2 , comprising at least 9 nucleotides. 
     
     
         5 . A DNA according to  claim 4 , comprising the sequences in SEQ ID NO:6 and/or in SEQ ID NO:7. 
     
     
         6 . A polyclonal or monoclonal antibody, chimeric antibody, fully humanized antibody, anti-anti-Id antibody or fragment thereof which was prepared using a polypeptide according to  claim 1 . 
     
     
         7 . In an immunoassay for the detection of polypeptide in biological fluids using an antibody against said polypeptide, the improvement wherein said polypeptide in Cari and said antibody is an antibody according to  claim 6 . 
     
     
         8 . The method according to  claim 7 , for diagnostic purposes. 
     
     
         9 . A method of producing Cari or an isoform, mutein, allelic variant, fragment, fusion protein or derivative thereof comprising the generation of a transgenic animal having a DNA sequence according to  claim 2  and capable of expressing the polypeptide encoded by said DNA sequence into the body fluids of the animal, and isolating the polypeptide produced from the body fluids of the animal. 
     
     
         10 . A method of gene therapy for treatment of an inflammatory disease selected from multiple sclerosis with primary oligodendrogliopathy, autoimmune uveoretinitis, diabetes, lupus, autoimmune myocarditis I, HCV mediated chronic hepatitis, chronic gastritis, mixed connective tissue disease (MCTD), Crohn's disease, or ulcerative colitis, comprising introducing a DNA sequence in accordance with  claim 2  so as to induce the expression of the polypeptide encoded thereby at a desired site in a human patient in need. 
     
     
         11 . A method according to  claim 10 , wherein the mutein is Cari D600E. 
     
     
         12 . A method according to  claim 10 , wherein the fragment comprises amino acid residues from 414 to 437 of Cari (SEQ ID NO:4). 
     
     
         13 . A method according to  claim 10 , wherein the fragment comprises amino acid residues from 422 to 437 of Cari (SEQ ID NO:5). 
     
     
         14 . A method according to  claim 10 , wherein the antisense oligonucleotide comprises the sequence on SEQ ID NO:6 and/or in SEQ ID NO:7. 
     
     
         15 . A method for the treatment of an inflammatory disease selected from multiple sclerosis with primary oligodendrogliopathy, autoimmune uveoretinitis, diabetes, lupus, autoimmune myocarditis I, HCV mediated chronic hepatitis, chronic gastritis, mixed connective tissue disease (MCTD), Crohn's disease, or ulcerative colitis, comprising regulation of endogenous Cari or Cari inhibitor, by targeting at a desired site in a human patient in need, a vector having a DNA regulatory sequence functional in cells for enabling endogenous gene activation of an endogenous gene encoding the Cari polypeptide (SEQ ID NO:3), said vector further including targeting sequences corresponding to portions of said endogenous gene such that, after a step of homologous recombination, said regulating sequence will be appropriately targeted to allow said endogenous gene activation. 
     
     
         16 . A method according to  claim 15 , for endogenous gene activation of Cari. 
     
     
         17 . A method according to  claim 15 , for endogenous gene activation of a Cari inhibitor. 
     
     
         18 . A method for down-regulation of a caspase, in situations where excessive cell death by apoptosis occurs, comprising administering an effective amount of a DNA according to  claim 2 . 
     
     
         19 . The method according to claim  418 , wherein the DNA comprises SEQ ID NO:6 and or SEQ ID NO:7. 
     
     
         20 . A method for down-regulation of a caspase, in situations where excessive cell death by apoptosis occurs, comprising administering an effective amount of an antibody according to  claim 6 . 
     
     
         21 . A method for up-regulation of a caspase activity and increase of apoptosis in situations where excessive cell death is required, comprising administering an effective amount of a polypeptide according to  claim 1 . 
     
     
         22 . A method for up-regulation of a caspase activity and increase of apoptosis in situations where excessive cell death is required, comprising administering an effective amount of a DNA according to  claim 2 . 
     
     
         23 . A method for up-regulation of a caspase activity and increase of apoptosis in situations where excessive cell death is required, comprising administering an effective amount of an antibody according to  claim 6 . 
     
     
         24 . A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to  claim 6 . 
     
     
         25 . A pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of endogenous Cari activity for the treatment or prevention of a disease in which the activity of Cari is involved. 
     
     
         26 . A method for the isolation, identification and cloning of another polypeptide of the same class of Cari comprising the use of a DNA according to  claim 2  to screen a DNA library. 
     
     
         27 . A method for the isolation, identification and cloning of another polypeptide of the same class of Cari from a sample selected from body fluids, cell extracts and DNA expression libraries, comprising co-immunoprecipitating a caspase and the polypeptide using antibodies specific for the caspase. 
     
     
         28 . A method for the isolation, identification and cloning of another polypeptide of the same class of Cari, comprising affinity purification of such polypeptide in a sample using an antibody according to  claim 6 . 
     
     
         29 . A method according to  claim 28 , wherein the sample is selected from body fluids, cell extracts and DNA expression libraries. 
     
     
         30 . A method for isolating polypeptides or factors involved in intracellular signaling processes, comprising the use of a polypeptide according to  claim 1  as the prey or the bait in the yeast two-hybrid procedure. 
     
     
         31 . A method according to  claim 30 , wherein the mutein is Cari D600E mutant. 
     
     
         32 . A method according to  claim 30 , wherein the fragment comprises amino acid residues from 414 to 437 of Cari (SEQ ID NO:4). 
     
     
         33 . A method according to  claim 30 , wherein the fragment comprises amino acid residues from 422 to 437 of Cari (SEQ ID NO:5). 
     
     
         34 . A method for isolating a polypeptide or factor involved in intracellular signaling processes, from samples selected from cell extracts human fluids and expression libraries, comprising co-immunoprecipitating Cari and the polypeptides or factors involved in intracellular signaling using an antibody according to  claim 6 . 
     
     
         35 . A method for screening for a peptide or a small molecule antagonist to Cari, comprising high throughput screening and selection of such molecules able to inhibit the interaction of Cari to pro-caspase-8 or a mutein, isoform, allelic variant, fragment, fusion protein or derivative thereof according to  claim 1 . 
     
     
         36 . A method according to  claim 35 , wherein the mutein is Cari D600E. 
     
     
         37 . A method according to  claim 35 , wherein the fragment comprises amino acid residues from 414 to 437 of Cari (SEQ ID NO:4). 
     
     
         38 . A method according to  claim 35 , wherein the fragment comprises amino acid residues from 422 to 437 of Cari (SEQ ID NO:5). 
     
     
         39 . A method for screening for a peptide or a small molecule antagonist to Cari, comprising high throughput screening and selection of such molecules able to inhibit apoptosis enhanced by Cari according to  claim 1 . 
     
     
         40 . A method according to  claim 39 , wherein the mutein is Cari D600E. 
     
     
         41 . A method of treatment and/or prevention of a disorder selected from, multiple sclerosis with primary oligodendrogliopathy, autoimmune uveoretinitis, diabetes, lupus, autoimmune myocarditis I, HCV mediated chronic hepatitis, chronic gastritis, mixed connective tissue disease (MCTD), Crohn's disease, ulcerative colitis and cancer, comprising administering to a patient in need thereof a pharmaceutically effective amount of a Cari polypeptide or a mutein, isoform, allelic variant, fragment, fusion protein or derivative thereof according to  claim 1 . 
     
     
         42 . A method according to  claim 41 , wherein the mutein is Cari D600E mutant. 
     
     
         43 . A method according to  claim 41 , wherein the fragment comprises amino acid residues from 414 to 437 of Car (SEQ ID NO:4). 
     
     
         44 . A method according to  claim 41 , wherein the fragment comprises amino acid residues from 422 to 437 of Car (SEQ ID NO:5). 
     
     
         45 . A method of treatment and/or prevention of a disorder selected from, multiple sclerosis with primary oligodendrogliopathy, autoimmune uveoretinitis, diabetes, lupus, autoimmune myocarditis I, HCV mediated chronic hepatitis, chronic gastritis, mixed connective tissue disease (MCTD), Crohn's disease, ulcerative colitis and cancer comprising administering to a patient in need thereof a pharmaceutically effective amount of a DNA according  claim 2 . 
     
     
         46 . A method of treatment and/or prevention of a disorder selected from, multiple sclerosis with primary oligodendrogliopathy, autoimmune uveoretinitis, diabetes, lupus, autoimmune myocarditis I, HCV mediated chronic hepatitis, chronic gastritis, mixed connective tissue disease (MCTD), Crohn's disease, ulcerative colitis and cancer comprising administering to a patient in need thereof a pharmaceutically effective amount of an antibody according to  claim 6 . 
     
     
         47 . A vector having a DNA regulatory sequence functional in cells for enabling endogenous gene activation of an endogenous gene encoding an inhibitor of the Cari polypeptide of SEQ ID NO:3, said vector further including targeting sequences corresponding to portions of said endogenous gene such that, after a step of homologous recombination, said regulating sequence will be appropriately targeted to allow said endogenous gene activation. 
     
     
         48 . The method according to  claim 18 , wherein the caspase is caspase-8. 
     
     
         49 . The method according to  claim 20 , wherein the caspase is caspase-8. 
     
     
         50 . The method according to  claim 18 , wherein the apoptosis is induced by the TNF receptor signaling pathway. 
     
     
         51 . The method according to  claim 20 , wherein the apoptosis is induced by the TNF receptor signaling pathway. 
     
     
         52 . The method according to  claim 21 , wherein the apoptosis is induced by the TNF receptor signaling pathway. 
     
     
         53 . The method according to  claim 22 , wherein the caspase is caspase-8. 
     
     
         54 . The method according to  claim 23 , wherein the caspase is caspase-8.