Soluble TNF Receptors and Their Use in Treatment of Disease

Abstract

The present invention relates to tumor necrosis factor (TNF) antagonists and corresponding nucleic acids derived from tumor necrosis factor receptors (TNFRs) and their use in the treatment of inflammatory diseases. These proteins are soluble secreted decoy receptors that bind to TNF and prevent TNF from signaling to cells. In particular, the proteins are mammalian TNFRs that lack exon 7 and which can bind TNF and can act as a TNF antagonist.

1 . An isolated protein capable of binding tumor necrosis factor (TNF), said protein having a sequence comprising the amino acids encoded by a cDNA derived from a mammalian tumor necrosis factor receptor (TNFR) gene, wherein the cDNA comprises in 5′ to 3′ contiguous order,
 the codon encoding the first amino acid after the cleavage point of the signal sequence of said gene through exon 6 of said gene and exon 8 of said gene through exon 10 of said gene; or 
 the codon encoding the first amino acid of the open reading frame of said gene through exon 6 of said gene and exon 8 of said gene through exon 10 of said gene. 
 
     
     
         2 . The protein of  claim 1 , wherein said TNF is TNF-α. 
     
     
         3 . The protein of  claim 1 , wherein said protein contains at least one processing, chemical, or post-translational modification, and wherein said modification is selected from the group consisting of acetylation, acylation, amidation, ADP-ribosylation, glycosylation, methylation, pegylation, prenylation, phosphorylation, or cholesterol conjugation. 
     
     
         4 . The protein of  claim 1 , wherein said receptor is TNFR1. 
     
     
         5 . The protein of  claim 4 , wherein said receptor is human TNFR1. 
     
     
         6 . The protein of  claim 1 , wherein said receptor is TNFR2. 
     
     
         7 . The protein of  claim 6 , wherein said receptor is human TNFR2. 
     
     
         8 . The protein of  claim 1 , wherein the sequence of said protein comprises a sequence selected from the group consisting of SEQ ID No: 6, amino acids 30-417 of SEQ ID No: 6, SEQ ID No: 8, amino acids 30-416 of SEQ ID No: 8, SEQ ID No: 10, amino acids 23-435 of SEQ ID No: 10, SEQ ID No: 12, and amino acids 23-448 of SEQ ID No: 12. 
     
     
         9 . A pharmaceutical composition comprising the protein of any one of  claim 1  or  7 , in admixture with a pharmaceutically acceptable carrier. 
     
     
         10 . A composition comprising the purified protein of  claim 1 . 
     
     
         11 . A method of treating an inflammatory disease or condition which comprises administering the pharmaceutical composition of  claim 9  to a subject for a time and in an amount effective to reduce the activity of TNF. 
     
     
         12 . The method of  claim 11 , wherein said disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (Crohn's disease or ulcerative colitis), hepatitis associated with hepatitis A virus, hepatitis associated with hepatitis B virus, hepatitis associated with hepatitis C virus, hepatitis associated with ischemia/reperfusion, sepsis, alcoholic liver disease, and non-alcoholic steatosis. 
     
     
         13 . An isolated nucleic acid derived from a mammalian tumor necrosis factor receptor (TNFR) gene and encoding a protein capable of binding tumor necrosis factor (TNF), wherein the cDNA of said protein comprises in 5′ to 3′ contiguous order,
 the codon encoding the first amino acid after the cleavage point of the signal sequence of said gene through exon 6 of said gene and exon 8 of said gene through exon 10 of said gene; or 
 the codon encoding the first amino acid of the open reading frame of said gene through exon 6 of said gene and axon 8 of said gene through axon 10 of said gene. 
 
     
     
         14 . The nucleic acid of  claim 13 , wherein said receptor is TNFR1. 
     
     
         15 . The nucleic acid of  claim 14 , wherein said receptor is human TNFR1. 
     
     
         16 . The nucleic acid of  claim 13 , wherein said receptor is TNFR2. 
     
     
         17 . The nucleic acid of  claim 16 , wherein said receptor is human TNFR2. 
     
     
         18 . The nucleic acid of  claim 13 , wherein the sequence of said protein comprises a sequence selected from the group consisting of SEQ ID No: 6, amino acids 30-417 of SEQ ID No: 6, SEQ ID No: 8, amino acids 30-416 of SEQ ID No: 8, SEQ ID No: 10, amino acids 23-435 of SEQ ID No: 10, SEQ ID No: 12, and amino acids 23-448 of SEQ ID No: 12. 
     
     
         19 . The nucleic acid of  claim 16 , wherein the sequence of said nucleic acid comprises a sequence selected from the group consisting of nucleotides 1-1251 of SEQ ID No: 5, nucleotides 88-1251 of SEQ ID No: 5, nucleotides 1-1248 of SEQ ID No: 7, nucleotides 88-1248 of SEQ ID No: 7, nucleotides 1-1305 of SEQ ID No: 9, nucleotides 67-1305 of SEQ ID No: 9, nucleotides 1-1344 of SEQ ID No: 11, and nucleotides 67-1344 of SEQ ID No: 11. 
     
     
         20 . An expression vector comprising the nucleic acid of any one of  claim 13  or  17  operably linked to a regulatory sequence. 
     
     
         21 . A method of increasing the level of a TNF antagonist in a mammal which comprises transforming cells of said mammal with the expression vector of  claim 20  to thereby express said TNF antagonist, wherein said vector drives expression of said TNFR. 
     
     
         22 . The method of  claim 21 , wherein said mammal is a human. 
     
     
         23 . The method of  claim 22 , wherein said human is an individual having an inflammatory disease or condition. 
     
     
         24 . The method of  claim 21 , wherein said expression vector is a plasmid, or a virus. 
     
     
         25 . The method of  claim 21 , wherein said cells are transformed in vivo. 
     
     
         26 . The method of  claim 21 , wherein said cells are transformed ex vivo. 
     
     
         27 . The method of  claim 21 , wherein said expression vector comprises a tissue specific promoter. 
     
     
         28 . The method of  claim 27 , wherein said tissue specific promoter is derived from a hepatocyte or a macrophage. 
     
     
         29 . The method of  claim 21 , wherein said cells are selected from the group consisting of hepatocytes, hematopoietic cells, spleen cells, and muscle cells. 
     
     
         30 . A cell transformed with the expression vector of  claim 20 . 
     
     
         31 . The cell of  claim 30 , wherein said cell is a mammalian cell, an insect cell, or a microbial cell. 
     
     
         32 . A process for producing a protein capable of binding tumor necrosis factor (TNF) which comprises culturing the cell of  claim 30  under conditions suitable to express said protein, and recovering said protein. 
     
     
         33 . The process of  claim 32 , wherein said cell is a mammalian cell, an insect cell, or a microbial cell. 
     
     
         34 . A pharmaceutical composition comprising the nucleic acid of  claim 13 , in admixture with a pharmaceutically acceptable carrier. 
     
     
         35 . A method of treating an inflammatory disease or condition which comprises administering the expression vector of  claim 20  to a subject for a time and in an amount sufficient to reduce TNF activity. 
     
     
         36 . The method of  claim 35 , wherein said TNF activity is TNF-α activity. 
     
     
         37 . The method of  claim 35 , wherein said disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (Crohn's disease or ulcerative colitis), hepatitis associated with hepatitis A virus, hepatitis associated with hepatitis B virus, hepatitis associated with hepatitis C virus, hepatitis associated with ischemia/reperfusion, sepsis, alcoholic liver disease, and non-alcoholic steatosis. 
     
     
         38 . A method of treating an inflammatory disease or condition which comprises administering one or more splice switching oligomers (SSOs) to a subject for a time and in an amount to reduce the activity of TNF, wherein said one or more SSOs are capable of altering the splicing of a pre-mRNA encoding a mammalian tumor necrosis factor receptor 2 (TNFR2) to increase production of a protein capable of binding tumor necrosis factor (TNF),
 wherein said protein has a sequence comprising the amino acids encoded by a cDNA derived from a gene for said receptor, wherein the cDNA comprises in 5′ to 3′ contiguous order,
 the codon encoding the first amino acid after the cleavage point of the signal sequence of said gene through exon 6 of said gene and exon 8 of said gene through exon 10 of said gene; or 
 the codon encoding the first amino acid of the open reading frame of said gene through exon 6 of said gene and exon 8 of said gene through exon 10 of said gene. 
   
     
     
         39 . The method of  claim 38 , wherein said receptor is a human TNFR2. 
     
     
         40 . The method of  claim 38 , wherein said disease or condition is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (Crohn's disease or ulcerative colitis), hepatitis associated with hepatitis A virus, hepatitis associated with hepatitis B virus, hepatitis associated with hepatitis C virus, hepatitis associated with ischemia/reperfusion, sepsis, alcoholic liver disease, and non-alcoholic steatosis. 
     
     
         41 . The method of  claim 39 , wherein the sequence of said SSO comprises at least 8 nucleotides which are complementary to a contiguous sequence from SEQ ID No: 13. 
     
     
         42 . The method of  claim 41  wherein the sequence of said SSO comprises a sequence selected from the group consisting of SEQ ID Nos: 14, 30, 46, 70, 71, 72, and 73, and subsequences thereof at least 8 nucleotides. 
     
     
         43 . The method of  claim 42 , wherein the sequence of said SSO comprises a sequence selected from the group consisting of SEQ ID Nos: 14-61. 
     
     
         44 . The method of  claim 38 , wherein said administration is parenteral, topical, oral, rectal, or pulmonary. 
     
     
         45 . A method of increasing the production of a protein capable of binding tumor necrosis factor (TNF), in a cell, which comprises administering one or more splice switching oligomers (SSOs) to said cell,
 wherein said protein has a sequence comprising the amino acids encoded by a cDNA derived from a mammalian tumor necrosis factor receptor 2 (TNFR2) gene, wherein the cDNA comprises in 5′ to 3′ contiguous order,
 the codon encoding the first amino acid after the cleavage point of the signal sequence of said gene through exon 6 of said gene and exon 8 of said gene through exon 10 of said gene; or 
 the codon encoding the first amino acid of the open reading frame of said gene through exon 6 of said gene and exon 8 of said gene through exon 10 of said gene, and 
   wherein said one or more SSOs are capable of altering the splicing of a pre-mRNA encoding said receptor to increase production of said protein.   
     
     
         46 . The method of  claim 45 , wherein said method is performed in vivo. 
     
     
         47 . The method of  claim 45 , wherein said receptor is a human TNFR2. 
     
     
         48 . The method of  claim 47 , wherein said SSO comprises at least 8 nucleotides which are complementary to a contiguous sequence from SEQ ID No: 13. 
     
     
         49 . The method of  claim 48 , wherein the sequence of said SSO comprises a sequence selected from the group consisting of SEQ ID Nos: 14, 30, 46, 70, 71, 72, and 73, and subsequences thereof at least 8 nucleotides. 
     
     
         50 . The method of  claim 49 , wherein the sequence of said SSO comprises a sequence selected from the group consisting of SEQ ID Nos: 14-61. 
     
     
         51 . A splice switching oligomer (SSO) comprising at least 8 nucleotides, said SSO capable of altering the splicing of a pre-mRNA encoding a mammalian tumor necrosis factor receptor 2 (TNFR2) to produce a protein capable of binding tumor necrosis factor (TNF),
 wherein said protein has a sequence comprising the amino acids encoded by a cDNA derived from a gene for said receptor, wherein the cDNA comprises in 5′ to 3′ contiguous order,
 the codon encoding the first amino acid after the cleavage point of the signal sequence of said gene through exon 6 of said gene and exon 8 of said gene through exon 10 of said gene; or 
 the codon encoding the first amino acid of the open reading frame of said gene through exon 6 of said gene and exon 8 of said gene through exon 10 of said gene. 
   
     
     
         52 . The SSO of  claim 51 , wherein said receptor is a human TNFR2. 
     
     
         53 . The SSO of  claim 52 , which comprises at least 8 nucleotides which are complementary to a contiguous sequence from SEQ ID No: 13. 
     
     
         54 . The SSO of  claim 53 , wherein the sequence of said SSO comprises a sequence selected from the group consisting of SEQ ID Nos: 14, 30, 46, 70, 71, 72, and 73, and subsequences thereof at least 8 nucleotides. 
     
     
         55 . The SSO of  claim 54 , wherein the sequence of said SSO comprises a sequence selected from the group consisting of SEQ ID Nos: 14-61. 
     
     
         56 . A pharmaceutical composition comprising the SSO of  claim 51  and a pharmaceutically acceptable carrier.