High affinity tcr proteins and methods

Abstract

T cell receptors (TCRS) that have higher affinity for a ligand than wild type TCRs are provided. These high affinity TCRs are formed by mutagenizing a T cell receptor protein coding sequence to generate a variegated population of mutants of the T cell receptor protein coding sequence; transforming the T cell receptor mutant coding sequence into yeast cells; inducing expression of the T cell receptor mutant coding sequence on the surface of yeast cells; and selecting those cells expressing T cell receptor mutants that have higher affinity for the peptide/MHC ligand than the wild type T cell receptor protein. The high affinity TCRs can be used in place of an antibody or single chain antibody.

1 . A method for cloning the gene for a high affinity TCR mutant into a system that allows expression of the mutant on the surface of T cells comprising:
 mutating TCRs to create high affinity TCR mutants;   cloning said TCR mutants into a vector;   transfecting the vector into T cells;   expressing the high affinity TCR mutant on the surface of T cells.   
     
     
         2 . The method of  claim 1 , further comprising:
 selecting those T cells that are activated by a peptide/MHC ligand more than the wild type.   
     
     
         3 . The method of  claim 1 , wherein the transfected/infected T cells are used for recognition of selected peptide-bearing MHC cells. 
     
     
         4 . T cells made by the method of  claim 1 . 
     
     
         5 . A method for cloning the gene for a high affinity TCR mutant into a system that allows expression of the mutant on the surface of T cells comprising the steps of:
 mutating TCRs to create high affinity TCR mutants which exhibit a dissociation constant for their cognate ligand of at least about 10 7  M −1 ;   cloning said TCR mutants into a vector;   transfecting the vector into T cells; and   expressing the high affinity TCR mutant on the surface of T cells.   
     
     
         6 . The method of  claim 5 , wherein the transfected T cells are used for recognition of selected peptide-bearing MHC cells. 
     
     
         7 . The method of  claim 5  wherein the high affinity TCR mutants carry one or more mutations in a CDR. 
     
     
         8 . The method of  claim 7  wherein the high affinity TCR mutants carry one or more mutations in CDR3α or CDR3β. 
     
     
         9 . A method for cloning the gene for a high affinity TCR mutant into a system that allows expression of the mutant on the surface of T cells comprising the steps of:
 mutating TCRs to create high affinity TCR mutants carrying one or more mutations in a CDR;   cloning said TCR mutants into a vector;   transfecting the vector into T cells; and   expressing the high affinity TCR mutant on the surface of T cells.   
     
     
         10 . The method of  claim 9 , wherein the transfected T cells are used for recognition of selected peptide-bearing MHC cells. 
     
     
         11 . The method of  claim 9  wherein the high affinity TCR mutants carry one or more mutations in CDR3α or CDR3β. 
     
     
         12 . T cells made by the methods of  claim 5 . 
     
     
         13 . A DNA sequence encoding a mutant high affinity TCR exhibiting a dissociation constant of greater than about 10 7  M −1  for its cognate ligand. 
     
     
         14 . The DNA sequence of  claim 13  wherein the TCR mutant exhibits a dissociation constant between about 10 7  and 10 10  M −1  for its cognate ligand. 
     
     
         15 . The DNA sequence of  claim 13  wherein the TCR mutant carries one or more mutations in a CDR. 
     
     
         16 . The DNA sequence of  claim 15  wherein the TCR mutant carries one or more mutations in CDR3α or CDR3β. 
     
     
         17 . A DNA sequence encoding a mutant high affinity TCR carrying one or more mutations in CDR. 
     
     
         18 . The DNA sequence of  claim 17  carrying one or mutations in CDR3α or CDR3β. 
     
     
         19 . T cells made by the methods of  claim 9 .