Small molecular weight TNF receptor multimeric molecule

Abstract

The present invention relates to a receptor molecule which binds to TNF comprising all or a functional portion of the extracellular domain (ECD) of two or more TNF-Rs linked via one or more polypeptide linkers. The receptor can further comprise a signal peptide of a secreted protein, such as the signal peptide of the extracellular domain of the TNF-R or the signal peptide of a cytokine. The invention also relates to isolated DNA encoding a receptor molecule which binds to TNF, comprising two or more sequences encoding all or a functional portion of the ECD of TNF-Rs linked via one or more sequences encoding a polypeptide linker. The invention further relates to a method of making a construct which expresses all or a functional portion of the ECD of two or more TNF-Rs linked via one or more polypeptide linkers and cells which express the construct. The invention also relates to a method of inhibiting the biological activity of TNF in a host comprising administering to the host an effective amount of a receptor molecule of the present invention. The invention further relates to receptor molecules which bind cytokines that bind to receptor molecules comprising more than one subunit.

1 - 23 . (canceled)  
     
     
         24 . A receptor molecule which binds to tumor necrosis factor comprising all or a functional portion of a first extracellular domain of tumor necrosis factor receptor bound via a peptide bond to a first polypeptide linker, wherein said first polypeptide linker is bound via a peptide bond to all or a functional portion of a second extracellular domain of tumor necrosis factor receptor bound via a peptide bond to a second polypeptide linker, wherein said second polypeptide linker is bound via a peptide bond to all or a functional portion of a third extracellular domain of tumor necrosis factor receptor, wherein the first and second polypeptide linkers are each from about 10 to about 30 amino acid residues in length, and wherein the receptor molecule is capable of binding to a tumor necrosis factor trimer in a stoichiometric ratio of almost 1:1.  
     
     
         25 . The receptor molecule of  claim 24 , wherein the first extracellular domain, the second extracellular domain, and the third extracellular domain are independently selected from the group consisting of (i) the extracellular domain of a p75 tumor necrosis factor receptor, (ii) the extracellular domain of a p55 tumor necrosis factor receptor or (iii) functional portions of (i) or (ii).  
     
     
         26 . The receptor molecule of  claim 24  further comprising a signal peptide of a secreted protein.  
     
     
         27 . The receptor molecule of  claim 25 , wherein the first, second and third extracellular domains are the same.  
     
     
         28 . Isolated DNA encoding a receptor molecule according to  claim 24 .  
     
     
         29 . A method of making a construct which expresses the receptor molecule of  claim 24  comprising the steps of: 
 a) obtaining a first vector which expresses an extracellular domain of a first tumor necrosis factor receptor and a signal peptide of a secreted protein;    b) obtaining a second vector which expresses an extracellular domain of a second tumor necrosis factor receptor;    c) obtaining a third vector which expresses an extracellular domain of a third tumor necrosis factor receptor; and    d) ligating the first vector of (a) to the second vector of (b) and litigating the second vector of (b) to the third vector of (c) using a coding sequence for a first polypeptide linker so that the first vector of (a) is linked to the second vector of (b) using the coding sequence for the first polypeptide linker and using a second coding sequence for a second polypeptide linker so that the second vector of (b) is linked to the third vector of (c) using the second coding sequence for the second polypeptide linker, resulting in a construct which expresses the receptor molecule of  claim 24 .    
     
     
         30 . A cell which expresses a receptor molecule according to  claim 24  comprising a nucleic acid encoding such receptor molecule.  
     
     
         31 . A method of inhibiting the biological activity of tumor necrosis factor in a subject comprising administering to a subject a tumor necrosis factor-inhibiting amount of a receptor molecule according to  claim 24 .  
     
     
         32 . A method of treating a tumor necrosis factor-related disease in a subject in need thereof comprising administering to the subject a tumor necrosis factor-inhibiting amount of a receptor molecule according to  claim 24 .  
     
     
         33 . The method of  claim 32 , wherein the tumor necrosis factor-related disease is selected from the group consisting of an autoimmune disease, an inflammatory bowel disease, a bacterial infection, a viral infection, a parasitic infection, a malignancy, and a neurodegenerative disease.  
     
     
         34 . The method of  claim 32 , wherein the tumor necrosis factor-related disease is selected from the group consisting of rheumatoid arthritis, septic shock, cerebral malaria, inflammatory bowel disease, multiple sclerosis, allograft rejection, host versus graft disease, neoplastic pathology and endotoxemic response.  
     
     
         35 . The method of  claim 32 , wherein the tumor necrosis factor-related disease is rheumatoid arthritis.  
     
     
         36 . The receptor molecule of  claim 24 , wherein the first extracellular domain, the second extracellular domain, and the third extracellular domain are of human origin and wherein the first and second polypeptide linkers are polyglycine linker sequences.