Transposition-mediated identification of specific binding or functional proteins

Abstract

The method disclosed herein describes a novel technology offering unparalleled efficiency, flexibility, utility and speed for the discovery and optimization of polypeptides having desired binding specificity and/or functionality, including antigen-binding molecules such as antibodies and fragments thereof, for desired functional and/or binding phenotypes. The novel method is based on transposable constructs and diverse DNA libraries cloned into transposable vectors and their transfection into host cells by concomitant transient expression of a functional transposase enzyme. This ensures an efficient, stable introduction of the transposon-based expression vectors into vertebrate host cells in one step, which can then be screened for a desired functional or binding phenotype of the expressed proteins, after which the relevant coding sequences for the expressed proteins, including antibodies and fragments thereof, can be identified by standard cloning and DNA sequencing techniques.

1 - 20 . (canceled) 
     
     
         21 . A method of obtaining a polypeptide having a desired binding specificity or functionality, comprising:
 (i) generating a diverse collection of polynucleotides encoding polypeptides having different binding specificities or functionalities, wherein said polynucleotides each comprise a sequence encoding a polypeptide, said sequence disposed between first and second inverted terminal repeat sequences that are recognized by and functional with a least one transposase enzyme;   (ii) introducing the diverse collection of polynucleotides of (i) into host cells;   (iii) expressing at least one transposase enzyme functional with said inverted terminal repeat sequences in said host cells so that said diverse collection of polynucleotides is integrated into the host cell genomes to provide a host cell population that expresses said polypeptides having different binding specificities or functionalities;   (iv) screening said host cells to identify a host cell expressing a polypeptide having a desired binding specificity or functionality; and   (v) isolating the polynucleotide sequence encoding said polypeptide identified in step (iv) from said host cell.   
     
     
         22 . The method according to  claim 21 , wherein said polynucleotides comprise a sequence encoding:
 a) a ligand-binding domain of a receptor or a target-binding domain of a binding molecule,   b) an antigen-binding domain of an antibody,   c) a V H  or V L  region of an antibody or an antigen-binding fragment thereof,   d) an antibody V H  region and an antibody V L  region,   e) a full-length immunoglobulin heavy chain or light chain or an antigen-binding fragment thereof, or   f) a single-chain Fv or a Fab domain.   
     
     
         23 . The method according to  claim 22 , wherein generating said diverse collection of polynucleotides comprises subjecting V region gene sequences to PCR under mutagenizing conditions. 
     
     
         24 . The method according to  claim 21 , wherein step (ii) comprises introducing into said host cells polynucleotides comprising sequences encoding:
 a) immunoglobulin V H  or V L  regions or antigen-binding fragments thereof, wherein said V H  and V L  region sequences are encoded on separate vectors,   b) full-length immunoglobulin heavy or light chains, or antigen-binding fragments thereof, wherein said full-length heavy and light chain sequences are encoded on separate vectors,   c) an antibody V H  and V L  chains encoded on the same vector, or   d) a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain encoded on the same vector.   
     
     
         25 . The method according to  claim 21 , wherein said expressing step (iii) comprises introducing into said host cells an expression vector encoding a transposase enzyme that recognizes and is functional with an least one inverted terminal repeat sequence, wherein said transposase enzyme is transiently expressed in said host cells. 
     
     
         26 . The method according to  claim 21 , wherein said screening step (iv) comprises: magnetic activated cell sorting (MACS), fluorescence activated cell sorting (FACS), panning against molecules immobilized on a solid surface, selection for binding to cell-membrane associated molecules incorporated into a cellular, natural or artificially reconstituted lipid bilayer membrane, or high-throughput screening of individual cell clones in a multi-well format for a desired functional or binding phenotype. 
     
     
         27 . The method according to  claim 21 , wherein said step (v) of isolating the polynucleotide sequence encoding the polypeptide having a desired binding specificity or functionality comprises genomic or RT-PCR amplification or next-generation deep sequencing. 
     
     
         28 . The method according to  claim 21 , wherein
 a) said inverted terminal repeat sequences are from the PiggyBac transposon system or the Sleeping Beauty transposon system, and/or   b) step (iii) comprises introducing into said host cells a vector comprising a sequence encoding a functional PiggyBac transposase or Sleeping Beauty transposase.   
     
     
         29 . The method according to  claim 21 , wherein said inverted terminal repeat sequences are recognized by and functional with at least one transposase selected from the group consisting of: PiggyBac, Sleeping Beauty, Frog Prince, Himar1, Passport, Minos, hAT, Tol1, Tol2, Ac/Ds, PIF, Harbinger, Harbinger3-DR, and Hsmar1. 
     
     
         30 - 34 . (canceled) 
     
     
         35 . A vector or set of vectors, comprising a sequence encoding a V H  or V L  region of an antibody, or antigen-binding portion thereof, disposed between inverted terminal repeat sequences that are recognized by and functional with at least one transposase enzyme. 
     
     
         36 . The vector or set of vectors of  claim 35 , wherein said V H  or V L  region sequences encode a sequence derived from a human anti-TNF alpha antibody. 
     
     
         37 . The vector or set of vectors of  claim 36 , wherein the V H  or V L  regions are encoded by separate transposable vectors. 
     
     
         38 . The vector or set of vectors of  claim 36 , wherein the antibody sequences are from D2E7. 
     
     
         39 . The vector or set of vectors of  claim 35 , wherein a first vector having the V H  sequence comprises inverted terminal repeat sequences that are recognized by a different transposase enzyme than the inverted terminal repeat sequences in a second vector that has the V L  sequence. 
     
     
         40 . A host cell comprising a vector or set of vectors according to  claim 35 . 
     
     
         41 . The host cell of  claim 40 , wherein the host cell is mammalian. 
     
     
         42 . The host cell of  claim 41 , wherein the host cell is human or rodent. 
     
     
         43 . The host cell of  claim 42 , wherein the host cell is a lymphoid cell. 
     
     
         44 . The host cell of  claim 43 , wherein the host cell is selected from: B cell, progenitor B cells, precursor B cells, Abelson-Murine Leukemia virus transformed progenitor B cells or precursor B cells, and early, immunoglobulin-null EBV transformed human proB or preB cells. 
     
     
         45 - 47 . (canceled) 
     
     
         48 . A method for generating a population of host cells capable of expressing polypeptides having different binding specificities or functionalities, comprising:
 (i) generating a diverse collection of polynucleotides comprising sequences encoding polypeptides having different binding specificities or functionalities, wherein said polynucleotides comprise a sequence encoding a polypeptide having a binding specificity or functionality disposed between inverted terminal repeat sequences that are recognized by and functional with a least one transposase enzyme; and   (ii) introducing said diverse collection of polynucleotides into the host.