Mutant Forms of Fas Ligand and Uses Thereof

Abstract

The invention provides for DNA encoding Fas ligand muteins and chimeras and the proteins encoded thereby. The invention further includes the use of DNA and vectors to produce transformed cells expressing the mutant or chimeric Fas ligand. When the Fas ligand of the invention is a non cleavable form, the cells expressing the Fas ligand are useful in vitro for identifying Fas expressing cells or in vivo for reducing populations of Fas expressing cells. Thus, in other embodiments, the present invention is also directed to a method for treating a patient, for example a mammal, for autoimmune disease or transplant rejection by administering a Fas ligand therapeutic agent. The therapeutic agent is a polypeptide, a polynucleotide encoding the polypeptide or a small molecule. The polypeptides include full-length Fas ligand polypeptide, or a biologically active variant, derivative, portion, fusion or peptide thereof.

1 . A method for determining the presence in a sample of cells expressing Fas, the method comprising the steps of:
 a) providing a transformed cell having a recombinant Fas ligand surface bound thereto, said surface bound, recombinant Fas ligand being resistant to cleavage;   b) contacting said transformed cell with a sample containing cells suspected of having surface bound Fas thereon to obtain a cell mixture;   c) allowing said cell bound Fas to bind to said surface bound recombinant Fas ligand; and   d) observing said cell mixture for rosette formation or clumping, whereby the binding of cells from said sample to said transformed cell indicates the presence of Fas on the surface of cells in said sample.   
     
     
         2 . The method of  claim 1 , wherein said recombinant Fas ligand is selected from the group consisting of a Fas ligand deletion mutein and a Fas ligand chimera,
 said Fas ligand deletion mutein being a deletion mutein of pro-Fas ligand lacking a continuous segment of 10 to 17 amino acid residues beginning at residue position 130 of pro-Fas ligand (SEQ ID NO: 12),   said Fas ligand chimera being a fusion protein having an amino acid sequence that corresponds to the fusion product of the carboxy terminus of a transmembrane domain of a cell surface protein other than Fas ligand,   said transmembrane domain being substantially non-cleavable from a cell membrane by proteinases or convertases and being fused to the amino terminus of the extracellular domain of Fas ligand,   said extracellular domain lacking a continuous segment of 10 to 17 amino acid residues beginning at about residue position 130 of pro-Fas ligand (SEQ ID NO: 12).   
     
     
         3 . The method of  claim 2 , wherein said Fas ligand mutein has an amino acid sequence that is the same as the amino acid sequence in SEQ ID NO: 6 or SEQ ID NO: 10. 
     
     
         4 . The method of  claim 3 , wherein said Fas ligand mutein has an amino acid sequence that is the same as the amino acid sequence in SEQ ID NO: 6. 
     
     
         5 . The method of  claim 3 , wherein said Fas ligand mutein has an amino acid sequence that is the same as the amino acid sequence in SEQ ID NO: 10. 
     
     
         6 . The method of  claim 2 , wherein said Fas ligand chimera has an amino acid sequence that corresponds to the fusion product of the carboxy terminus of the transmembrane domain of CD30 ligand fused to the amino terminus of the extracellular domain of Fas ligand, said extracellular domain lacking a continuous segment of 10 to 17 amino acid residues beginning at about residue position 130 of pro-Fas ligand. 
     
     
         7 . The method of  claim 6 , wherein said Fas ligand chimera has an amino acid sequence corresponding to the fusion product of the carboxy terminus of residues 1-86 of CD30 ligand fused to the amino terminus of the extracellular domain of Fas ligand, said extracellular domain beginning 10 to 17 amino acid residues beyond about residue position 130 and extending to residue position 281 of pro-Fas. 
     
     
         8 . The method of  claim 2 , wherein said Fas ligand chimera has an amino acid sequence corresponding to the fusion product of the carboxy terminus of the transmembrane domain of CD40 ligand fused to the amino terminus of the extracellular domain of Fas ligand, said extracellular domain lacking a continuous segment of 10 to 17 amino acid residues beginning at about residue position 130 of pro-Fas ligand. 
     
     
         9 . The method of  claim 8 , wherein said Fas ligand chimera has an amino acid sequence corresponding to the fusion product of the carboxy terminus of residues 1-107 of CD40 ligand fused to the amino terminus of the extracellular domain of Fas ligand, said extracellular domain beginning 10 to 17 amino acid residues beyond about residue position 130 and extending to residue position 281 of pro-Fas ligand. 
     
     
         10 . The method of  claim 1 , wherein said recombinant Fas ligand is a polypeptide comprising:
 a transmembrane domain of a cell surface protein; and   a Fas ligand portion, wherein said Fas ligand portion comprises a sequence at least 90% homologous to SEQ ID NO:7, and wherein the Fas ligand portion binds Fas;   wherein said polypeptide does not contain 17 contiguous amino acid residues starting at position 130 of SEQ ID NO: 12.   
     
     
         11 . The method of  claim 10 , wherein said transmembrane domain is an uncleavable transmembrane portion of a transmembrane protein other than pro-Fas ligand. 
     
     
         12 . The method of  claim 10 , wherein said Fas ligand portion comprises SEQ ID NO:7. 
     
     
         13 . The method of  claim 10 , wherein the transmembrane domain is a transmembrane domain of a polypeptide selected from the group consisting of a membrane-bound antibody, a membrane bound viral antigen, and a member of the tumor necrosis factor receptor superfamily of polypeptides. 
     
     
         14 . The method of  claim 13 , wherein the transmembrane domain comprises a transmembrane domain of a tumor necrosis factor receptor superfamily polypeptide. 
     
     
         15 . The method of  claim 14 , wherein the tumor necrosis factor receptor superfamily polypeptide is selected from the group consisting of tumor necrosis factor receptor, CD30, nerve growth factor receptor, CD27, CD40, CD120a, CD120b, lymphotoxin beta receptor, and TRAIL receptor. 
     
     
         16 . The method of  claim 10 , wherein the transmembrane domain comprises a transmembrane domain of CD30 ligand. 
     
     
         17 . The method of  claim 10 , wherein the transmembrane domain comprises a transmembrane domain of CD40 ligand.