Use of nanoexpression to interrogate antibody repertoires

Abstract

Disclosed herein are methods and compositions for using nanoexpression to interrogate antigen specificity within antibody repertoires. Also disclosed herein is an antibody display system composition comprising one or more recombinant nucleic acid sequences comprising a first nucleic acid sequence encoding a heavy chain variable region sequence or fragment thereof operatively linked to a first identification region sequence and a second nucleic acid sequence encoding a light chain variable region sequence or fragment thereof operatively linked to a second identification region sequence.

1 . An antibody display system composition comprising one or more recombinant nucleic acid sequences comprising a first nucleic acid sequence encoding a heavy chain variable region sequence or fragment thereof operatively linked to a first identification region sequence and a second nucleic acid sequence encoding a light chain variable region sequence or fragment thereof operatively linked to a second identification region sequence. 
     
     
         2 . The composition of  claim 1 , wherein the first and second identification region sequences are distinct. 
     
     
         3 . The composition of  claim 1 , wherein the first and second identification region sequences are identical. 
     
     
         4 . The composition of  claim 1 , wherein the one or more recombinant nucleic acid sequences comprises a Fab sequence. 
     
     
         5 . The composition of  claim 1 , wherein the one or more recombinant nucleic acid sequences comprises a scFv sequence. 
     
     
         6 . The composition of  claim 1 , wherein the antibody display system composition is a phagemid. 
     
     
         7 . The composition of  claim 1 , wherein the antibody display system composition is a ribosome, cell, mRNA, mammalian cell, bacteria, or yeast cell. 
     
     
         8 . An antibody display system library comprising a plurality of the antibody display system composition of  claim 1 . 
     
     
         9 . (canceled) 
     
     
         10 . A method of annotating an antibody repertoire, comprising:
 obtaining a first dataset, wherein the first dataset comprises sequence data for a plurality of heavy and light chain antibody sequence pairs each from a distinct single sample, wherein each sequence of each pair is operatively linked to an identification region sequence, and wherein each identification region sequence is unique relative to the other identification region sequences within the plurality;   creating an antibody display system using the dataset, wherein the antibody display system comprises a plurality of components;   binding the antibody display system to an antigen;   determining a second dataset comprising binding data and sequence data based on the binding step; and   annotating the sequence data of the first dataset with the data of the second dataset.   
     
     
         11 .- 20 . (canceled) 
     
     
         21 . A method of producing an antibody repertoire comprising native pairs of heavy and light chain variable regions, comprising:
 synthesizing polynucleotides from a single sample, wherein the polynucleotides comprise a B cell variable immunoglobulin region sequence and an identification region sequence;   generating a first dataset, wherein the first dataset comprises sequence data for a plurality of heavy and light chain antibody sequence pairs each from a distinct single sample, wherein each sequence of each pair is operatively linked to the identification region sequence;   expressing an antibody, or an immunologically functional fragment thereof, from the polynucleotides, wherein the antibody comprises a native pair of heavy and light chain sequences;   binding the antibody or immunologically functional fragment to antigen; and   selecting sequences from the first dataset for producing the antibody repertoire based on the binding step.   
     
     
         22 . The method of  claim 21 , wherein the single sample is a single B cell. 
     
     
         23 . The method of  claim 21 , wherein the binding data is affinity data. 
     
     
         24 . The method of  claim 21 , wherein the binding data is antigen-specificity data. 
     
     
         25 . The method of  claim 21 , wherein the binding data is determined using an ELISA assay. 
     
     
         26 . The method of  claim 21 , wherein the antigen is a recombinant protein, a cell, a subcellular fraction, a bacterium, fungi, protozoa, or a tissue. 
     
     
         27 . The method of  claim 21 , wherein the sequence data is determined using sequencing. 
     
     
         28 . The method of  claim 21 , wherein the sequence data is determined using next generation sequencing (NGS).