Fusion proteins, uses thereof and processes for producing same

Abstract

This invention provides fusion proteins comprising consecutive amino acids which beginning at the amino terminus of the protein correspond to consecutive amino acids present in (i) a cytomegalovirus human MHC-restricted peptide, (ii) a first peptide linker, (iii) a human β-2 microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi) a third peptide linker, (vii) a variable region from a heavy chain of a scFv fragment of an antibody, and (viii) a variable region from a light chain of such scFv fragment, wherein the consecutive amino acids which correspond to (vii) and (viii) are bound together directly by a peptide bond or by consecutive amino acids which correspond to a fourth peptide linker, wherein the antibody from which the scFv fragment is derived specifically binds to mesothelin. This invention provides nucleic acid constructs encoding same, processes for producing same, compositions, and uses thereof.

1 . A fusion protein comprising consecutive amino acids which, beginning at the amino terminus of the protein, correspond to consecutive amino acids present in (i) a cytomegalovirus human MHC-restricted peptide, (ii) a first peptide linker, (iii) a human β-2 microglobulin, (iv) a second peptide linker, (v) a HLA-A2 chain of a human MHC class I molecule, (vi) a third peptide linker, (vii) a variable region from a heavy chain of a scFv fragment of an antibody, and (viii) a variable region from a light chain of such scFv fragment, wherein the consecutive amino acids which correspond to (vii) and (viii) are bound together directly by a peptide bond or by consecutive amino acids which correspond to a fourth peptide linker and the scFv fragment is derived from an antibody which specifically binds to mesothelin. 
     
     
         2 . The fusion protein of  claim 1 , wherein the first peptide linker has the amino acid sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:6). 
     
     
         3 . The fusion protein of  claim 1 , wherein the second peptide linker has the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO:8). 
     
     
         4 . The fusion protein of  claim 1 , wherein the third peptide linker has the amino acid sequence ASGG (SEQ ID NO:10). 
     
     
         5 . The fusion protein of  claim 1 , wherein the fourth peptide linker has the amino acid sequence GVGGSGGGGSGGGGS (SEQ ID NO:19). 
     
     
         6 . The fusion protein of  claim 1 , wherein the cytomegalovirus human MHC-restricted peptide has the amino acid sequence NLVPMVATV (SEQ ID NO:4). 
     
     
         7 . The fusion protein of  claim 1 , wherein the sequence of the consecutive amino acids corresponding to (vii), followed by the fourth peptide linker, followed by (viii) is set forth in SEQ ID NO:12. 
     
     
         8 . The fusion protein of  claim 1 , wherein the consecutive amino acids have the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         9 . A composition comprising the fusion protein of  claim 1  and a carrier. 
     
     
         10 . The composition of  claim 9  wherein the fusion protein is present in the composition in a therapeutically effective amount and the carrier is a pharmaceutically acceptable carrier. 
     
     
         11 . A nucleic acid construct comprising a nucleic acid sequence encoding the fusion protein of  claim 1 . 
     
     
         12 . The nucleic acid construct of  claim 11 , wherein said nucleic acid sequence is as set forth in SEQ ID NO:1. 
     
     
         13 . A vector comprising the nucleic acid construct of  claim 11 . 
     
     
         14 . An expression vector comprising the nucleic acid construct of  claim 11  and a promoter operatively linked thereto. 
     
     
         15 . A transformed cell comprising the vector of  claim 14 . 
     
     
         16 . An isolated preparation of bacterially-expressed inclusion bodies comprising over 30 percent by weight of the fusion protein of  claim 1 . 
     
     
         17 . A process for producing a fusion protein comprising culturing the transformed cell of  claim 15 , so that the fusion protein is expressed, and recovering the fusion protein so expressed. 
     
     
         18 . A method of killing a tumor cell which expresses mesothelin on its surface, the method comprising contacting the tumor cell with the fusion protein of  claim 1  in an amount effective to initiate a CTL-mediated immune response against the tumor cell so as to thereby kill the tumor cell.