Single chain class i major histocompatibility complexes, constructs encoding same and methods of generating same

Abstract

Provided are methods of generating a functional mammalian single chain MHC class I complex in prokaryotic expression systems and a host cell transformed with expression construct(s) capable of expressing a functional human single chain MHC class I complex capable of presenting specific antigenic peptides restricted to specific CTL clones.

1 . A method of producing a plurality of identical MHC class I-antigenic peptide complexes being recognizable by one CTL clone comprising:
 (a) expressing in bacteria, a single chain MHC class I polypeptide comprising a functional human β-2 microglobulin amino acid sequence covalently linked upstream of a functional human MHC class I heavy chain amino acid sequence; and   (b) isolating said single chain MHC class I polypeptide; and   (c) refolding said single chain MHC class I polypeptide in a presence of an antigenic peptide capable of binding said single chain MHC class I polypeptide, thereby obtaining the plurality of identical MHC class I-antigenic peptide complexes, said plurality of MHC class I-antigenic peptide complexes being recognizable by one CTL clone.   
     
     
         2 . The method of  claim 1 , further comprising the step of:
 (d) isolating said MHC class I-antigenic peptide complexes via size exclusion chromatography.   
     
     
         3 . The method of  claim 1 , wherein step (a) is effected such that said single chain MHC class I polypeptide forms inclusion bodies in said bacteria. 
     
     
         4 . The method of  claim 3 , wherein said step of isolating said polypeptide further comprises denaturing said inclusion bodies so as to release protein molecules therefrom. 
     
     
         5 . The method of  claim 1 , further comprising reducing said single chain MHC class I polypeptide prior to step (c). 
     
     
         6 . The method of  claim 1 , wherein said refolding is effected under renaturation conditions. 
     
     
         7 . The method of  claim 6 , wherein said renaturation conditions comprise an oxidizing agent. 
     
     
         8 . The method of  claim 6 , wherein said renaturation conditions comprise an oxidized glutathione. 
     
     
         9 . The method of  claim 7 , wherein said renaturation conditions further comprise arginine. 
     
     
         10 . The method of  claim 8 , wherein said renaturation conditions further comprise arginine. 
     
     
         11 . A nucleic acid construct encoding a recombinant human MHC class I molecule, comprising a first polynucleotide encoding a functional human β-2 microglobulin, being translationally fused upstream of a second polynucleotide encoding a functional human MHC class I heavy chain. 
     
     
         12 . The nucleic acid construct of  claim 11 , further comprising an in-frame tag sequence encoding a peptide capable of being enzymatically modified to include a binding entity. 
     
     
         13 . The nucleic acid construct of  claim 12 , wherein said peptide is a biotin protein ligase Bir A enzyme recognition sequence. 
     
     
         14 . The nucleic acid construct of  claim 11 , further includes an in-frame linker polynucleotide sequence encoding a linker peptide interposed between said first and said second polynucleotides. 
     
     
         15 . A bacterial host cell being transformed with the nucleic acid construct of  claim 11 . 
     
     
         16 . A bacterial host cell being transformed with the nucleic acid construct of  claim 12 . 
     
     
         17 . A method of producing a plurality of identical MHC class I-antigenic peptide complexes being recognizable by one CTL clone comprising:
 (a) isolating said recombinant human MHC class I molecule from the bacterial host cell of  claim 16 ; and   (c) refolding said recombinant human MHC class I molecule in a presence of an antigenic peptide capable of binding said recombinant human MHC class I molecule, thereby obtaining the plurality of identical MHC class I-antigenic peptide complexes, said plurality of MHC class I-antigenic peptide complexes being recognizable by one CTL clone.   
     
     
         18 . The method of  claim 17 , further comprising assembling said plurality of identical MHC class I-antigenic complexes into a multimer using a ligand which binds to said binding entity. 
     
     
         19 . The method of  claim 18 , wherein said binding entity is a Bir A enzyme recognition sequence and whereas said ligand is avidin or streptavidin.