Use of a tag to enrich polypeptides libraries

Abstract

The invention is directed to production of chimeric antibodies using display screening methods. The invention is based in part on two related but self-sufficient improvements in conventional display methods. The first improvement provides methods of enriching conventional display libraries for members displaying more than one copy of a polypeptide prior to affinity screening of such libraries with a target of interest. These methods can achieve diverse populations in which the vast majority of members retaining full-length coding sequences encode polypeptides having specific affinity for the target. In a second aspect, the invention provides methods of subcloning nucleic acids encoding displayed polypeptides of enriched libraries from a display vector to an expression vector without the need for clonal isolation of individual members. These methods can be used to produce polyclonal libraries of chimeric antibodies for use, e.g., as diagnostic or therapeutic reagents.

1 - 56 . (canceled)  
     
     
         57 . A method of selecting a member of a library having specific affinity for a target, wherein members of the library are capable of displaying a protein comprising a tag common to different members of the library and a polypeptide to be screened varying between members of the library, and some but not all of the library members display the protein; the method comprising the steps of: 
 a) contacting the library with a receptor having affinity for the tag and selecting members bound thereto, thereby obtaining a first selected pool of members enriched for members displaying the protein; and    b) contacting the first selected pool of members with a target and selecting a population of members which bind to the target; 
 wherein said contacting steps (a) and (b) select members that bind the target at the polypeptide and the receptor at the tag.  
   
     
     
         58 . The method according to  claim 57 , wherein the library is contacted with a target and then with the receptor.  
     
     
         59 . The method according to  claim 57 , wherein library members further comprise a second tag common to members of the library and two or more pools are selected using two or more receptors.  
     
     
         60 . The method according to  claim 57 , wherein the polypeptides of the library are antibody chains.  
     
     
         61 . The method according to  claim 60 , wherein the polypeptides comprise a V H  domain  
     
     
         62 . The method according to  claim 57 , wherein the polypeptides comprise V L .  
     
     
         63 . The method according to  claim 57 , wherein the members comprise a heavy and light chain, one of which is the polypeptide and the other is a binding partner  
     
     
         64 . The method according to  claim 57 , wherein the receptor is selected from the group consisting of a metal chelate ligands, a protein, a peptide, and an antibody  
     
     
         65 . The method according to  claim 57 , wherein some library members lack the protein due to proteolytic cleavage.  
     
     
         66 . The method according to  claim 57 , wherein sequences encoding the heavy and light chain are combined at the genetic level in a vector, and each chain is linked to a tag.  
     
     
         67 . The method of  claim 57 , wherein the library members are bacteriophage encoding the proteins.  
     
     
         68 . A method for selecting a functional polypeptide from a repertoire of polypeptides comprising functional and non-functional polypeptides, said functional polypeptides being those which bind to a target ligand at a first binding site and to the same generic ligand at a second binding site, said generic ligand having affinity for a structural feature common to all functional members of the repertoire, said non-functional polypeptides being unable to bind to a target or generic ligand, the method comprising the steps of: 
 a) contacting the repertoire with the generic ligand and selecting polypeptides bound thereto, thereby obtaining a first selected pool of polypeptides enriched for said functional polypeptides and comprising a reduced proportion of non-functional polypeptides; and    b) contacting the first selected pool of polypeptides with a target ligand and selecting a population of polypeptides which bind to the target ligand; 
 wherein said contacting steps (a) and (b) select polypeptides that bind target ligand at said first binding site and generic ligand at said second binding site.  
   
     
     
         69 . The method according to  claim 68 , wherein the repertoire of polypeptides is contacted with a target ligand and then with the generic ligand.  
     
     
         70 . The method according to  claim 68 , wherein two or more pools are selected using two or more generic ligands.  
     
     
         71 . The method according to  claim 70 , wherein two or more first selected pools are pooled after selection to produce a second selected pool of polypeptides enriched for functional polypeptides.  
     
     
         72 . The method according to  claim 68 , wherein two or more repertoires of polypeptides are contacted with generic ligands and first selected pools of polypeptides obtained from each repertoire are pooled to form a second selected pool of polypeptides.  
     
     
         73 . The method according to  claim 68 , wherein the polypeptides of the repertoire belong to immunoglobulin superfamily.  
     
     
         74 . The method according to  claim 73 , wherein the polypeptides comprise antibody or T-cell receptor polypeptides.  
     
     
         75 . The method according to  claim 74 , wherein the polypeptides comprise V H  or V beta  domains.  
     
     
         76 . The method according to  claim 74 , wherein the polypeptides comprise V L  or V alpha.  domains.  
     
     
         77 . The method according to  claim 67 , wherein a repertoire of polypeptides comprising V H  or V beta  domains and a repertoire of polypeptides comprising V L  or V alpha  domains are contacted with generic ligands and the first selected pools of polypeptides obtained for each repertoire are pooled to form a second selected pool of polypeptides.  
     
     
         78 . The method according to  claim 57 , wherein the generic ligand is selected from the group consisting of a matrix of metallic ions, an organic compound, a protein, a peptide, a monoclonal antibody, a polyclonal antibody population, and a superantigen.  
     
     
         79 . A method for detecting one or more members of a repertoire of polypeptides previously selected according to  claim 67 , comprising binding said one or more members to the generic ligand and detecting bound ligand.  
     
     
         80 . The method according to  claim 68 , wherein said non-functional polypeptides comprises polypeptides selected from the group consisting of: improperly folded polypeptides, polypeptides comprising missense mutations, and polypeptides comprising frameshift mutations.  
     
     
         81 . The method according to  claim 71 , wherein said first selected pools of polypeptides obtained from each repertoire are each contacted with different target ligands prior to being pooled to form said second selected set of pooled polypeptides.  
     
     
         82 . The method according to  claim 71 , wherein sequences encoding said second selected pool of polypeptides are combined at the genetic level in a vector which encodes a polypeptide having binding sites for two generic ligands.  
     
     
         83 . The method according to  claim 81 , wherein sequences encoding said second selected pool of polypeptides are combined at the genetic level in a vector which encodes a polypeptide having binding sites for two generic ligands and two target ligands.  
     
     
         84 . The method according to  claim 79 , wherein the superantigen is Protein A or Protein L.  
     
     
         85 . The method according to  claim 72 , wherein said first selected pools of polypeptides obtained from each repertoire are each contacted with different target ligands prior to being pooled to form said second selected set of pooled polypeptides.  
     
     
         86 . The method according to  claim 77 , wherein said first selected pools of polypeptides obtained from each repertoire are each contacted with different target ligands prior to being pooled to form said second selected set of pooled polypeptides.  
     
     
         87 . The method according to  claim 72 , wherein sequences encoding said second selected set of pooled polypeptides are combined at the genetic level in a vector which encodes a polypeptide having binding sites for two generic ligands.  
     
     
         88 . The method according to  claim 77 , wherein sequences encoding said second selected set of pooled polypeptides are combined at the genetic level in a vector which encodes a polypeptide having binding sites for two generic ligands.