Nucleic Acid Molecules Encoding TNF-Alpha Ligand Polypeptides Having a CD154 Domain

Abstract

The present invention is directed to an isolated polynucleotide sequence encoding a chimeric TNFα, comprising a first nucleotide sequence encoding a domain or subdomain of a tumor necrosis factor ligand other than TNFα, wherein the encoded domain or subdomain replaces a cleavage site of native TNFα, and a second nucleotide sequence encoding a domain or subdomain of native TNFα that binds to a TNFα receptor. The encoded chimeric TNFα is significantly less susceptible to cleavage from the cellular surface and, as a result can increase the concentration of a ligand capable of binding to a TNFα receptor on the surface of a cell. The chimeric TNFα is therefore useful in methods for inducing apoptosis of a cell expressing a TNFα receptor, inducing activation of an immune system cell and treating neoplastic cells, by introducing into the cell of interest an isolated polynucleotide sequence encoding a chimeric TNFα that is expressed on the surface of the cell.

1 . A chimeric TNFα ligand polypeptide having a CD 154 Domain III and a TNFα Domain IV, wherein:
 (a) the Domain III lacks a metalloproteinase cleavage site present in CD154; and, 
 (b) the Domain IV binds to a TNF receptor; 
 wherein the chimeric polypeptide is more resistant to cell membrane cleavage into soluble TNFα than are human pro-TNFα and human pro-TNFα lacking the TACE mmp recognition site spanning Val77 and Pro88 of human TNFα, when expressed from a coding nucleic acid in HeLa, 293, A549, COLO205, HCT15, BT20 or HT1080 cells. 
 
     
     
         2 . The chimeric TNFα ligand polypeptide of  claim 1  further comprising Domain II fragment of CD154. 
     
     
         3 . The chimeric TNFα ligand polypeptide of  claim 1  further comprising Domain I of CD154. 
     
     
         4 . The chimeric TNFα ligand polypeptide of  claim 2  further comprising Domain I of CD154. 
     
     
         5 . The chimeric TNFα ligand polypeptide of  claim 1  further comprising a linker domain comprising at least one amino acid that links the CD 154 Domain III to the TNFα Domain IV. 
     
     
         6 . A process for producing a chimeric TNFα ligand polypeptide of  claim 1  comprising culturing a host cell containing the coding nucleic acid molecule under conditions suitable to effect expression of the polypeptide.