Novel TNF receptor death domain ligand proteins and inhibitors of ligand binding

Abstract

Novel TNF receptor death domain (“TNF-R1-DD”) ligand proteins are disclosed. Polynucleotides encoding the TNF-R1-DD ligand protein are also disclosed, along with vectors, host cells, and methods of making the TNF-R1-DD ligand protein. Pharmaceutical compositions containing the TNF-R1-DD ligand protein, methods of treating inflammatory conditions, and methods of inhibiting TNF-R death domain binding are also disclosed. Methods of identifying inhibitors of TNF-R death domain binding and inhibitors identified by such methods are also disclosed.

1 . A composition comprising an isolated polynucleotide encoding a protein having TNF-R1-DD ligand protein activity. 
   
   
       2 . The composition of  claim 1  wherein said polynucleotide is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 2 to nucleotide 1231;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:1;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:2;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:2; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).   
   
   
       3 . The composition of  claim 1  wherein said polynucleotide sequence is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 2 to nucleotide 415;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:3;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:4;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:4; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).   
   
   
       4 . A composition of  claim 1  wherein said polynucleotide is operably linked to an expression control sequence. 
   
   
       5 . A host cell transformed with a composition of  claim 4 . 
   
   
       6 . The host cell of  claim 5 , wherein said cell is a mammalian cell. 
   
   
       7 . A process for producing an TNF-R1-DD ligand protein, which comprises:
 (a) growing a culture of the host cell of  claim 5  in a suitable culture medium; and   (b) purifying the TNF-R1-DD ligand protein from the culture.   
   
   
       8 . A composition comprising a protein having TNF-R1-DD ligand protein activity. 
   
   
       9 . The composition of  claim 8  wherein said protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:2; and   (b) fragments of the amino acid sequence of SEQ ID NO:2;   
     said protein being substantially free from other mammalian proteins. 
   
   
       10 . The composition of  claim 8  wherein said protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:4; and   (b) fragments of the amino acid sequence of SEQ ID NO:4;   
     said protein being substantially free from other mammalian proteins. 
   
   
       11 . The composition of  claim 8  wherein said protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:6; and   (b) fragments of the amino acid sequence of SEQ ID NO:6;   
     said protein being substantially free from other mammalian proteins. 
   
   
       12 . The composition of  claim 8 , further comprising a pharmaceutically acceptable carrier. 
   
   
       13 . A composition comprising an antibody which specifically reacts with the TNF-R1-DD ligand protein of  claim 8 . 
   
   
       14 . A method of identifying an inhibitor of TNF-R death domain binding which comprises:
 (a) combining an TNF-R death domain protein with a composition of  claim 8 , said combination forming a first binding mixture;   (b) measuring the amount of binding between the TNF-R death domain protein and the TNF-R1-DD ligand protein in the first binding mixture;   (c) combining a compound with the TNF-R death domain protein and an TNF-R1-DD ligand protein to form a second binding mixture;   (d) measuring the amount of binding in the second binding mixture; and   (e) comparing the amount of binding in the first binding mixture with the amount of binding in the second binding mixture;   
     wherein the compound is capable of inhibiting TNF-R death domain binding when a decrease in the amount of binding of the second binding mixture occurs. 
   
   
       15 . The method of  claim 14  wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:2;   (b) fragments of the amino acid sequence of SEQ ID NO:2;   (c) the amino acid sequence of SEQ ID NO:4;   (d) fragments of the amino acid sequence of SEQ ID NO:4;   (e) the amino acid sequence of SEQ ID NO:6;   (f) fragments of the amino acid sequence of SEQ ID NO:6;   (g) the amino acid sequence of SEQ ID NO:8; and   (h) fragments of the amino acid sequence of SEQ ID NO:8.   
   
   
       16 . A method of preventing or ameliorating an inflammatory condition which comprises administering a therapeutically effective amount of a composition of  claim 12 . 
   
   
       17 . TNF-R1-DD ligand protein produced according to the method of  claim 7 . 
   
   
       18 . A method of inhibiting TNF-R death domain binding comprising administering a therapeutically effective amount of a composition of  claim 12 . 
   
   
       19 . A method of preventing or ameliorating an inflammatory condition which comprises administering to a mammalian subject a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of IGFBP-5 and fragments thereof having TNF-R1-DD ligand protein activity. 
   
   
       20 . A method of inhibiting TNF-R death domain binding comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of IGFBP-5 and fragments thereof having TNF-R1-DD ligand protein activity. 
   
   
       21 . A composition comprising an inhibitor identified according to the method of  claim 14 . 
   
   
       22 . The composition of  claim 21  further comprising a pharmaceutically acceptable carrier. 
   
   
       23 . A method of preventing or ameliorating an inflammatory condition comprising administering to a mammalian subject a therapeutically effective amount of the composition of  claim 22 . 
   
   
       24 . A method of inhibiting TNF-R death domain binding comprising administering to a mammalian subject a therapeutically effective amount of the composition of  claim 22 . 
   
   
       25 . A composition comprising a pharmaceutically acceptable carrier and a protein selected from the group consisting of IGFBP-5 and fragments thereof having TNF-R1-DD ligand protein activity. 
   
   
       26 . A method of identifying an inhibitor of TNF-R death domain binding which comprises:
 (a) transforming a cell with a first polynucleotide encoding an TNF-R death domain protein, a second polynucleotide encoding an TNF-R1-DD ligand protein, and at least one reporter gene, wherein the expression of the reporter gene is regulated by the binding of the TNF-R1-DD ligand protein encoded by the second polynucleotide to the TNF-R death domain protein encoded by the first polynucleotide;   (b) growing the cell in the presence of and in the absence of a compound; and   (c) comparing the degree of expression of the reporter gene in the presence of and in the absence of the compound;   
     wherein the compound is capable of inhibiting TNF-R death domain binding when a decrease in the degree of expression of the reporter gene occurs. 
   
   
       27 . The method of  claim 26  wherein the second polynucleotide is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:1 from nucleotide 2 to nucleotide 1231;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:1, which encodes a protein having TNF-R1-DD ligand protein activity;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:2;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:2 and having TNF-R1-DD ligand protein activity;   (e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:3 from nucleotide 2 to nucleotide 415;   (f) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:3, which encodes a protein having TNF-R1-DD ligand protein activity;   (g) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:4;   (h) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:4 and having TNF-R1-DD ligand protein activity;   (i) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:5 from nucleotide 2 to nucleotide 559;   (j) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:5, which encodes a protein having TNF-R1-DD ligand protein activity;   (k) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:6;   (l) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:6 and having TNF-R1-DD ligand protein activity;   (m) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:7 from nucleotide 57 to nucleotide 875;   (n) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:7, which encodes a protein having TNF-R1-DD ligand protein activity;   (o) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:8;   (p) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:8 and having TNF-R1-DD ligand protein activity; and   (q) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(p), which encodes a protein having TNF-R1-DD ligand protein activity.   
   
   
       28 . The method of  claim 26  wherein the cell is a yeast cell. 
   
   
       29 . The composition of  claim 1  wherein said polynucleotide sequence is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 2 to nucleotide 931;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:9;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:10;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ED NO: 10; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).   
   
   
       30 . The composition of  claim 1  wherein said polynucleotide sequence is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 2 to nucleotide 1822;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:11;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:12;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).   
   
   
       31 . The composition of  claim 8  wherein said protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:10; and   (b) fragments of the amino acid sequence of SEQ ID NO:10;   
     said protein being substantially free from other mammalian proteins. 
   
   
       32 . The composition of  claim 8  wherein said protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:12; and   (b) fragments of the amino acid sequence of SEQ ID NO:12;   
     said protein being substantially free from other mammalian proteins. 
   
   
       33 . The method of  claim 14  wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:10;   (b) fragments of the amino acid sequence of SEQ ID NO:10;   (c) the amino acid sequence of SEQ ID NO:12; and   (d) fragments of the amino acid sequence of SEQ ID NO:12.   
   
   
       34 . The method of  claim 26  wherein the second polynucleotide is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:9 from nucleotide 2 to nucleotide 931;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:9, which encodes a protein having TNF-R1-DD ligand protein activity;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO: 10;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:10 and having TNF-R1-DD ligand protein activity;   (e) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:11 from nucleotide 2 to nucleotide 1822;   (f) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:11, which encodes a protein having TNF-R1-DD ligand protein activity;   (g) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:12; and   (h) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:12 and having TNF-R1-DD ligand protein activity; and   (i) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(h), which encodes a protein having TNF-R1-DD ligand protein activity.   
   
   
       35 . The composition of  claim 1  wherein said polynucleotide sequence is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 3 to nucleotide 2846;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:13;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:14;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:14; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).   
   
   
       36 . The composition of  claim 8  wherein said protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:14; and   (b) fragments of the amino acid sequence of SEQ ID NO:14;   
     said protein being substantially free from other mammalian proteins. 
   
   
       37 . The method of  claim 14  wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:14; and   (b) fragments of the amino acid sequence of SEQ ID NO:14.   
   
   
       38 . The method of  claim 26  wherein the second polynucleotide is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:13 from nucleotide 3 to nucleotide 2846;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:13, which encodes a protein having TNF-R1-DD ligand protein activity;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:14;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:14 and having TNF-R1-DD ligand protein activity; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d), which encodes a protein having TNF-R1-DD ligand protein activity.   
   
   
       39 . The composition of  claim 1  wherein said polynucleotide sequence is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 326 to nucleotide 5092;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:15;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:16;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:16; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).   
   
   
       40 . The composition of  claim 8  wherein said protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:16; and   (b) fragments of the amino acid sequence of SEQ ID NO:16;   
     said protein being substantially free from other mammalian proteins. 
   
   
       41 . The method of  claim 14  wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:16; and   (b) fragments of the amino acid sequence of SEQ ID NO:16.   
   
   
       42 . The method of  claim 26  wherein the second polynucleotide is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:15 from nucleotide 326 to nucleotide 5092;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:15, which encodes a protein having TNF-R1-DD ligand protein activity;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:16;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:16 and having TNF-R1-DD ligand protein activity; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d), which encodes a protein having TNF-R1-DD ligand protein activity.   
   
   
       43 . The composition of  claim 1  wherein said polynucleotide sequence is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 14 to nucleotide 2404;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:17;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO:18;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:18; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d).   
   
   
       44 . The composition of  claim 8  wherein said protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:18; and   (b) fragments of the amino acid sequence of SEQ ID NO: 18;   
     said protein being substantially free from other mammalian proteins. 
   
   
       45 . The method of  claim 14  wherein said TNF-R1-DD ligand protein comprises an amino acid sequence selected from the group consisting of:
 (a) the amino acid sequence of SEQ ID NO:18; and   (b) fragments of the amino acid sequence of SEQ ID NO:18.   
   
   
       46 . The method of  claim 26  wherein the second polynucleotide is selected from the group consisting of:
 (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:17 from nucleotide 14 to nucleotide 2404;   (b) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO:17, which encodes a protein having TNF-R1-DD ligand protein activity;   (c) a polynucleotide encoding an TNF-R1-DD ligand protein comprising the amino acid sequence of SEQ ID NO: 18;   (d) a polynucleotide encoding an TNF-R1-DD ligand protein comprising a fragment of the amino acid sequence of SEQ ID NO:18 and having TNF-R1-DD ligand protein activity; and   (e) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(d), which encodes a protein having TNF-R1-DD ligand protein activity.