Engineering t cell receptors

Abstract

The use of model T cell receptors (TCRs) as scaffolds for in vitro engineering of novel specificities is provided. TCRs with de novo binding to a specific peptide-major histocompatibility complex (MHC) product can be isolated by: 1) mutagenizing a T cell receptor protein coding sequence to generate a variegated population of mutants (a library), 2) selection of the library of TCR mutants with the specific peptide-MHC, using a process of directed evolution and a “display” methodology (e.g., yeast, phage, mammalian cell) and the peptide-MHC ligand. The process can be repeated to identify TCR variants with improved affinity for the selecting peptide-MHC ligand.

What is claimed is: 
     
         1 . A modified T cell receptor, or antigen binding fragment thereof, comprising a Vα and a Vβ derived from a wild type T cell receptor, wherein the Vα, the Vβ, or both, comprise a mutation in one or more complementarity determining regions (CDRs) relative to the wild type T cell receptor, wherein the modified T cell receptor binds to a non-cognate peptide-MHC not bound by the wild type T cell receptor. 
     
     
         2 . The modified T cell receptor of  claim 1 , wherein the wild type T cell receptor comprises the Vα amino acid sequence set forth in SEQ ID NO:1 and the Vβ amino acid sequence set forth in SEQ ID NO:2. 
     
     
         3 . The modified T cell receptor of  claim 2 , comprising a modified Vα comprising an amino acid sequence having at least 80% identity to the Vα amino acid sequence set forth in SEQ ID NO:1 and a modified Vβ comprising an amino acid sequence having at least 80% identity to the Vβ amino acid sequence set forth in SEQ ID NO:2, wherein the modified T cell receptor does not bind to the cognate peptide-MHC bound by the wild type T cell receptor. 
     
     
         4 . The modified T cell receptor of  claim 2 , wherein the modified T cell receptor comprises an amino acid substitution at one or more of CDR1α 31, CDR3α 98, CDR3β 99, CDR3α 97, CDR3β 102, CDR3α 99, CDR3β 100, CDR3β 101, CDR1α 32, CDR1β 30, CDR3β 98. 
     
     
         5 . The modified T cell receptor of  claim 2 , wherein the modified T cell receptor comprises the wild type amino acid at position CDR2α 51. 
     
     
         6 . The modified T cell receptor of  claim 5 , wherein the modified T cell receptor further comprises the wild type amino acid at position CDR1α 31. 
     
     
         7 . The modified T cell receptor of  claim 6 , wherein the modified T cell receptor further comprises the wild type amino acid at position CD1α 28 and CD1α52. 
     
     
         8 . The modified T cell receptor of  claim 1 , wherein the wild type T cell receptor is a single-chain T cell receptor A6-X15 comprising the amino acid sequence set forth in SEQ ID NO:3. 
     
     
         9 . The modified T cell receptor of  claim 1 , wherein the non-cognate peptide-MHC comprises Mart1:HLA.A2, SL9 HIV:HLA.A2, WT-1:HLA.A2, or SURV:HLA.A2. 
     
     
         10 . The modified T cell receptor of  claim 9 , comprising 1) a modified Vα region comprising an amino acid sequence having at least 90% identity to the Vα region of the amino acid sequence set forth in one of SEQ ID NOs:33, 41, or 42 and 2) a modified Vβ region comprising an amino acid sequence having at least 90% identity to the Vβ region of the amino acid sequence set forth in one of SEQ ID NOs:33, 41, or 42. 
     
     
         11 . The modified T cell receptor of  claim 10 , comprising the amino acid sequence set forth in one of SEQ ID NOs:33, 41, or 42. 
     
     
         12 . The modified T cell receptor of  claim 1 , wherein the modified T cell receptor is generated by in vitro selection of a yeast display library of mutant T cell receptors. 
     
     
         13 . The modified T cell receptor of  claim 1 , wherein the wild type T cell receptor is human. 
     
     
         14 . The modified T cell receptor of  claim 1 , wherein the modified T cell receptor is a single chain T cell receptor. 
     
     
         15 . The modified T cell receptor of  claim 1 , wherein the wild type T cell receptor binds HLA-A2. 
     
     
         16 . A polypeptide encoding the modified T cell receptor of  claim 1 . 
     
     
         17 . A polynucleotide encoding the polypeptide of  claim 16 . 
     
     
         18 . A modified T cell receptor, or antigen binding fragment thereof, comprising a Vα and a Vβ derived from a wild type T cell receptor, wherein the Vα comprises amino acid residues 140 to 256 of SEQ ID NO:34, and wherein the Vβ comprises amino acid residues 1 to 122 of SEQ ID NO:34. 
     
     
         19 . A modified T cell receptor, or antigen binding fragment thereof, comprising a Vα and a Vβ derived from a wild type T cell receptor, wherein the Vα comprises amino acid residues 140 to 255 of SEQ ID NO:43, and wherein the Vβ comprises amino acid residues 1 to 122 of SEQ ID NO:43. 
     
     
         20 . A method for engineering a T cell receptor, or an antigen binding fragment thereof, with a desired specificity comprising:
 a) isolating a polynucleotide that encodes a wild type T cell receptor, or an antigen binding fragment thereof;   b) generating a library of mutant T cell receptors, or antigen binding fragments thereof, wherein the mutant T cell receptors, or antigen-binding fragment thereof, comprise a mutation in one or more complementarity determining regions relative to the wild type T cell receptor;   c) expressing the mutant T cell receptors in a surface display system; and   d) selecting mutant T cell receptors that bind to a non-cognate peptide-MHC.   
     
     
         21 . The method of  claim 20  wherein the wild type T cell receptor comprises the Vα amino acid sequence set forth in SEQ ID NO:1 and the Vβ amino acid sequence set forth in SEQ ID NO:2. 
     
     
         22 . The method of  claim 20  wherein the wild type T cell receptor is a single-chain T cell receptor A6-X15 comprising the amino acid sequence set forth in SEQ ID NO:3. 
     
     
         23 . The method of  claim 20  wherein the surface display system is a yeast display system. 
     
     
         24 . The method of  claim 20  wherein the non-cognate peptide-MHC is Mart1:HLA.A2, SL9 HIV:HLA.A2, WT-1:HLA.A2, or SURV:HLA.A2. 
     
     
         25 . The method of  claim 20 , further comprising a step of affinity maturation.