Modulator of tnf/ngf superfamily receptors and soluble oligomeric tnf/ngf superfamily receptors

Abstract

The present invention generally concerns novel proteins which bind to the intracellular domains of the p55 and p75 TNF-Rs and the FAS-R, which are capable of modulating the function of the p55 and p75 TNF-Rs and the FAS-R, and the DNA sequences which encode them. The present invention also concerns new soluble oligomeric TNF-Rs, oligomeric FAS-Rs and oligomeric receptors having a mixture of TNF-Rs and FAS-Rs. In addition, the present invention concerns methods of preparation and uses of all of the aforementioned.

1 . A protein capable of binding to an intracellular domain of a TNF or FAS receptor, selected from the group comprising the proteins 55.1, 55.3, 4, 65, 14v1, 16v1, 5.3, 75.16, E3, E15, E19, 230, F2, F9 and DD11, and biologically active analogs and derivatives thereof.  
     
     
         2 . A pharmaceutical composition for the modulation of the TNF- or FAS-R ligand effect on cells comprising, as active ingredient, a protein according to  claim 1 , or the protein p55IC, p55DD, FAS-IC or FAS-DD, its biologically active fragments, analogs, derivatives or mixtures thereof.  
     
     
         3 . A pharmaceutical composition according to  claim 2  for treating cells by induction therein of TNF-associated effects, comprising, as active ingredient, p55-IC, portions thereof, analogs and derivatives thereof, and a pharmaceutically acceptable carrier.  
     
     
         4 . A method of treating tumors, comprising administering the pharmaceutical composition according to  claim 3  to induce IL-8 expression and the subsequent killing of tumor cells.  
     
     
         5 . A pharmaceutical composition for modulating the TNF- or FAS-R ligand effect on cells comprising, as active ingredient, a recombinant animal virus vector encoding a protein capable of binding a cell surface receptor and encoding a protein according to  claim 1 , or the protein p55IC, p55DD, FAS-IC or FAS-DD, its biologically active fragments or analogs.  
     
     
         6 . Antibodies, or active fragments or derivatives thereof, specific for the protein, analogs or derivatives of the protein according to  claim 1 .  
     
     
         7 . A method for modulating the TNF or FAS-R ligand effect on cells carrying a TNF-R or a FAS-R comprising treating said cells with antibodies or active fragments or derivatives thereof, according to  claim 6 , said treating being by application of a suitable composition containing said antibodies, active fragments or derivatives thereof to said cells, wherein when the IC-binding proteins of said cells are exposed on the extracellular surface, said composition is formulated for extracellular application, and when said IC-binding proteins are intracellular said composition is formulated for intracellular application.  
     
     
         8 . A pharmaceutical composition for modulating the TNF or FAS-R ligand effect on cells comprising as active ingredient, an oligonucleotide sequence encoding an anti-sense sequence of the DNA sequence encoding a protein capable of binding to an intracellular domain of a receptor belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor superfamily.  
     
     
         9 . A method for the modulation of the TNF or FAS-R ligand effect on cells carrying a TNF-R or a FAS-R, comprising treating said cells with one or more proteins, analogs or derivatives selected from the group consisting of the proteins, analogs and derivatives according to  claim 1  and a protein being the p55IC, p55DD, FAS-IC or FAS-DD, analogs or derivatives thereof, all of said proteins being capable of binding to the intracellular domain and modulating the activity of said TNF-R or FAS-R, wherein said treating of said cells comprises introducing into said cells said one or more proteins, analogs or derivatives in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more proteins, analogs or derivatives 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.  
     
     
         10 . A method for modulating the TNF or FAS-R ligand effect on cells carrying a TNF-R or FAS-R comprising treating said cells with an oligonucleotide sequence selected from a sequence encoding an antisense sequence of at least part of the DNA sequence encoding a protein capable of binding to an intracellular domain of a receptor belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor superfamily, and a sequence encoding the antisense sequence of p55IC, p55DD, FAS-IC or FAS-DD, said oligonucleotide sequence being capable of blocking the expression of at least one of the TNF-R or FAS-R intracellular domain binding proteins.  
     
     
         11 . A method for treating tumor cells or HIV-infected cells or other diseased cells, comprising: 
 (a) constructing a recombinant animal virus vector carrying a sequence encoding a viral surface protein that is capable of binding to a specific tumor cell surface receptor or HIV-infected cell surface receptor or receptor carried by other diseased cells and a sequence encoding a protein selected from the proteins, analogs and derivatives capable of binding to one or more of the intracellular domains of one or more receptors belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) receptor superfamily, and the p55 TNF-R intracellular domain (p55IC), its “death domain” (p55DD), the intracellular domain of FAS-R (FAS-IC), or its “death domain” (FAS-DD), or a biologically active analog or derivative thereof, said protein, when expressed in said tumor, HIV-infected, or other diseased cell being capable of killing said cell; and    (b) infecting said tumor or HIV-infected cells or other diseased cells with said vector of (a).    
     
     
         12 . A method for inducing TNF-associated effects in cells or tissues comprising treating said cells with one or more proteins, analogs or derivatives thereof, said one or more proteins being selected from a protein being essentially all of the self-associating intracellular domain of the p55 TNF-R (p55-IC) or portions thereof capable of self-associating and inducing, in a ligand (TNF)-independent manner, said TNF effect in the cells, wherein said treating of the cells comprises introducing into said cells said one or more proteins, analogs or derivatives in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more proteins, analogs or derivatives 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 . A method for modulating the TNF or FAS-R ligand effect on cells comprising applying the ribozyme procedure in which a vector encoding a ribozyme sequence capable of interacting with a cellular mRNA sequence encoding a protein according to  claim 1  or a mRNA sequence encoding p55IC, p55DD, FAS-IC or FAS-DD, 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 protein or said p55IC, p55DD, FAS-IC or FAS-DD in said cells.  
     
     
         14 . A method for isolating and identifying proteins, capable of binding to the intracellular domain binding proteins according to  claim 1 , comprising applying the yeast two-hybrid procedure in which a sequence encoding said intracellular domain binding protein is carried by one hybrid vector and sequence from a cDNA or genomic DNA library are 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 intracellular domain binding protein.  
     
     
         15 . A soluble, oligomeric tumor necrosis factor receptor (TNF-R) comprising at least two self-associated fusion proteins, each fusion protein having (a) at its one end, a TNF binding domain selected from the extracellular domain of a TNF-R, analogs or derivatives thereof, said extracellular domain, analogs or derivatives thereof being incapable of deleterious self-association leading to interference of TNF binding or less than optimal TNF binding, and being able to bind TNF; and (b) at its other end, a self-associating domain selected from (i) essentially all of the intracellular domain of the p55 TNF-R (p55-IC), extending from about amino acid residue 206 to about amino acid residue 426 of SEQ ID NO:37; (ii) the death domain of the p55-IC extending from about amino acid residue 328 to about amino acid residue 426 of SEQ ID NO:37; (iii) essentially all of the intracellular domain of the FAS/APO1 receptor (FAS-IC); (iv) the death domain of FAS-IC; and (v) analogs, fractions or derivatives of any one of (i)-(iv) being capable of self-association, wherein said at least two self-associated proteins self-associate only at said ends (b), and have said ends (a) capable of binding to at least two TNF monomers, each end (a) capable of binding one TNF monomer; and salts and functional derivatives of said soluble, oligomeric TNF-R.  
     
     
         16 . A soluble, oligomeric TNF-R according to  claim 15 , comprising as said at least two ends (a) essentially all of the extracellular domain of the p55-R extending from about amino acid residue 1 to about amino acid residue 172 of SEQ ID NO:37, and as its at least two ends (b) essentially all of said death domain of the p55-IC.  
     
     
         17 . A soluble, oligomeric TNF-R according to  claim 15 , comprising as its two ends (a) analogs or derivatives of the extracellular domain of the p55-R, each of said analogs or derivatives being capable of binding one TNF monomer, and being incapable of self-association, and as its at least two ends (b) essentially all of said death domain of p55-IC.  
     
     
         18 . A soluble, oligomeric TNF-R according to  claim 15 , comprising as said at least two ends (a) essentially all of the extracellular domain of the p55-R extending from about amino acid residue 1 to about amino acid residue 172 of SEQ ID NO:37, and as its at least two ends, and as its at least two ends (b) essentially all of said death domain of FAS-IC.  
     
     
         19 . A soluble, oligomeric TNF-R according to  claim 15 , comprising as its at least two ends (a) analogs or derivatives of the extracellular domain of the p55-R, each of said analogs or derivatives being capable of binding one TNF monomer, and being incapable of self-association, and as its at least two ends (b) essentially all of said death domain of FAS-IC.  
     
     
         20 . A soluble, oligomeric receptor having affinity for both TNF and FAS-R ligand (mixed affinity receptor), comprising at least two self-associated fusion proteins, one of which fusion proteins is a TNF-specific TNF-R-derived protein of  claim 15;  and the other fusion protein is a soluble, oligomeric FAS/APO1 receptor (FAS-R) comprising at least two self-associated fusion proteins, each fusion protein having (a) at its one end, a FAS ligand binding domain selected from the extracellular domain of a FAS-R, analogs or derivatives thereof being incapable of self-associating and being able to bind FAS ligand; and (b) at its other end, a self-associating domain selected from (i) essentially all of the intracellular domain of the p55 TNF-R (p55-IC), extending from about amino acid residue 206 to about amino acid residue 426 of the SEQ ID NO:37; (ii) the death domain of the p55-IC extending from about amino acid residue 328 to about amino acid residue 426 of SEQ ID NO:37; (iii) essentially all of the intracellular domain of the FAS/APO1 receptor (FAS-IC); (iv) the death domain of FAS-IC; and (v) analogs or derivatives of any one of (i)-(iv) being capable of self-association, wherein said at least two self-associated proteins only self-associate at said ends (b) and have said ends (a) capable of binding to at least two FAS ligand monomers, each end (a) capable of binding one FAS ligand monomer; and salts and functional derivatives of said soluble, oligomeric FAS-R.  
     
     
         21 . A pharmaceutical composition comprising the mixed affinity receptor according to  claim 20  as an active ingredient, and a pharmaceutically acceptable carrier.  
     
     
         22 . A method of antagonizing the deleterious effects of TNF and FAS-R ligand, comprising administering the pharmaceutical composition according to  claim 21 .  
     
     
         23 . An expression vector comprising a fusion protein sequence encoding said fusion proteins of  claim 15 .  
     
     
         24 . A host cell containing a vector according to  claim 23  capable of expressing said fusion protein sequence.  
     
     
         25 . A pharmaceutical composition comprising the soluble, oligomeric TNF-R, salts or functional derivatives thereof, according to  claim 15 , as active ingredient, and a pharmaceutically acceptable carrier.  
     
     
         26 . A method of antagonizing the deleterious effect of TNF in conditions wherein an excess of TNF is formed endogenously or is exogenously administered, comprising administering the pharmaceutical composition according to  claim 25  to a subject in need thereof.  
     
     
         27 . A method for maintaining the prolonged beneficial effects of TNF in mammals, comprising administering the pharmaceutical composition according to  claim 25  in combination with TNF being exogenously administered.  
     
     
         28 . A soluble, oligomeric FAS/APO1 receptor (FAS-R) comprising at least two self-associated fusion proteins, each fusion protein having (a) at its one end, a FAS ligand binding domain selected from the extracellular domain of a FAS-R, analogs or derivatives thereof being incapable of self-associating and being able to bind FAS ligand; and (b) at its other end, a self-associating domain selected from (i) essentially all of the intracellular domain of the p55 TNF-R (p55-IC), extending from about amino acid residue 206 to about amino acid residue 426 of SEQ ID NO:37; (ii) the death domain of the p55-IC extending from about amino acid residue 328 to about amino acid residue 426 of SEQ ID NO:37; (iii) essentially all of the intracellular domain of the FAS/APO1 receptor (FAS-IC); (iv) the death domain of FAS-IC; and (v) analogs or derivatives of any one of (i)-(iv) being capable of self-association, wherein said at least two self-associated proteins only self-associate at said ends (b) and have said ends (a) capable of binding to at least two FAS ligand monomers, each end (a) capable of binding one FAS ligand monomer; and salts and functional derivatives of said soluble, oligomeric FAS-R.  
     
     
         29 . An expression vector comprising a fusion protein sequence encoding said fusion proteins of  claim 28 .  
     
     
         30 . A host cell containing a vector according to  claim 29  capable of expressing said fusion protein sequence.  
     
     
         31 . A pharmaceutical composition comprising the soluble, oligomeric FAS-R, salts or functional derivatives thereof or mixtures thereof, according to  claim 28  as active ingredient, and a pharmaceutically acceptable carrier.  
     
     
         32 . A method of antagonizing the deleterious effect of FAS ligand in conditions wherein an excess of FAS ligand is formed endogenously or is exogenously administered, comprising administering the pharmaceutical composition according to  claim 31  to a subject in need thereof.