Generating Targeted Sequence Diversity in Proteins

Abstract

Methods of generating sequence diversity in a protein, such as a ligand-binding protein, are provided. The methods comprise targeted introduction of two or more recombination signal sequences (RSSs) into the protein coding sequence and introduction of the modified protein coding sequence into a recombination-competent host cell, specifically a recombination-competent host cell that is capable of expressing at least RAG-1 and RAG-2, thereby allowing for recombination of the protein coding sequence and expression of variant proteins. Also provided are polynucleotides comprising a nucleic acid sequence encoding a target protein, such as a ligand-binding protein, and comprising two or more RSSs, and compositions and host cells comprising same.

What is claimed is: 
     
         1 .- 48 . (canceled) 
     
     
         49 . A method of generating a variant protein having increased affinity for an antigen, the method comprising:
 providing a plurality of host cells which each comprise a polynucleotide encoding an antibody, an antigen-binding domain of the antibody, or a T-cell receptor (TCR);   causing at least one double-stranded break within one or more portions of the polynucleotide which encode complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) or a combination of CDR1 and CDR2;   rejoining ends of the at least one double-stranded break with double-stranded break repair proteins comprising terminal deoxynucleotidyl transferase (TdT) to produce variant polynucleotides; and   expressing a library of variant proteins from the variant polynucleotides, and screening the library for variant proteins having increased affinity for the antigen relative to affinity of the antibody, the antigen-binding domain of the antibody, or the TCR to the antigen; thereby generating the variant protein having increased affinity for the antigen.   
     
     
         50 . The method of  claim 49 , wherein the at least one double-stranded break is a single double-stranded break within a portion of the polynucleotide encoding CDR1. 
     
     
         51 . The method of  claim 49 , wherein the at least one double-stranded break is a single double-stranded break within a portion of the polynucleotide encoding CDR2. 
     
     
         52 . The method of  claim 49 , wherein the at least one double-stranded break comprises a break in a portion of the polynucleotide encoding CDR1 and a break in a portion of the polynucleotide encoding CDR2. 
     
     
         53 . The method of  claim 49 , wherein the rejoining generates a change in the length of the variant protein having increased affinity for the antigen as compared to the antibody, the antigen-binding domain of the antibody, or the TCR. 
     
     
         54 . The method of  claim 53 , wherein the rejoining generates both composition and length diversity in the variant protein having increased affinity for the antigen as compared to the antibody, the antigen-binding domain of the antibody, or the TCR.