Molecular Display Method

Abstract

There is provided herein a method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule by screening using molecular display in conjunction with the sequencing of positive and negative selection pools from the screen.

1 . A method for identifying and/or recovering at least one genetically encoded affinity reagent specific for a target molecule, the method comprising:
 (a) providing a molecular display system which displays a library of potential genetically encoded affinity reagents;   (b) screening the library against the target molecule to produce positive and negative selection pools, preferably with multiple rounds of selection;   (c) sequencing genetically encoded affinity reagents in each of the positive and negative selection pools;   (d) identifying at least one sequence that is more abundant in the positive selection pool as compared to the negative selection pool; and   (e) recovering at least one clone corresponding to the sequence.   
     
     
         2 . The method of  claim 1 , wherein the display system is selected from the group consisting of phage display, bacterial display, yeast display, ribosome display and mRNA display. 
     
     
         3 . The method of  claim 2 , wherein the display system is phage display. 
     
     
         4 . The method of  claim 1 , wherein the sequencing comprises deep/high-throughput sequencing. 
     
     
         5 . The method of  claim 4 , wherein the deep sequencing comprises Illumina sequencing. 
     
     
         6 . The method of  claim 4 , wherein the deep sequencing comprises Ion semiconductor sequencing. 
     
     
         7 . The method of  claim 1 , wherein each of the affinity reagents in the library contain unique sequence tags and the sequencing identifies the unique sequence tags. 
     
     
         8 . The method of  claim 7 , wherein the at least one clone is recovered by annealing primers specific for the unique sequence tags. 
     
     
         9 . The method of  claim 1 , wherein the affinity reagents are selected from the group consisting of nucleic acid molecules and polypeptides. 
     
     
         10 . The method of  claim 9 , wherein the affinity reagents are antibodies or antigen-binding fragments thereof. 
     
     
         11 . The method of  claim 10 , wherein the affinity reagents are synthetic antibodies. 
     
     
         12 . The method of  claim 11 , wherein the library is a synthetic Fab library. 
     
     
         13 . The method of  claim 7 , wherein the library is a synthetic Fab library and the unique sequence tag is in the CDR H3 region. 
     
     
         14 . The method of  claim 1 , wherein the target molecule is an epitope of a protein. 
     
     
         15 . The method of  claim 1 , wherein the protein is a cell surface protein. 
     
     
         16 . The method of  claim 1 , wherein the screening is performed against the target molecule presented on a cell surface. 
     
     
         17 . The method of  claim 16 , wherein target molecule is presented on the cell surface of a mammalian cell. 
     
     
         18 . The method of  claim 1 , wherein the sequences identified are more abundant in the positive selection pool as compared to the negative selection pool by a factor of at least 2, and in increasing preferably at least 3, at least 4 and at least 5.