Compositions and methods for generating antigen-binding units

Abstract

The present invention provides non-single-chain antigen-binding units that is stabilized by leucine zipper sequences. The experimental design is particularly useful for generating and screening for Nsc Abus that remain the binding capabilities to their respective antigens within a cell. The present invention also provides recombinant polynucleotides, host cells and kits comprising the vectors. Further provided by the invention are methods of using the subject vectors.

1 - 42 . (canceled)  
     
     
         43 . A non-single-chain antigen-binding unit comprising: (a) a light (L) chain polypeptide comprising a light (L) chain variable region fused to a first leucine zipper sequence; (b) a heavy (H) chain polypeptide comprising a heavy (H) chain variable region fused to a second leucine zipper sequence; wherein the L chain and the H chain polypeptides dimerize to form an antigen-binding site through an interaction between the first and second leucine zipper sequences.  
     
     
         44 . A recombinant polynucleotide comprising a coding sequence that encodes the L chain variable region of  claim 43 .  
     
     
         45 . The recombinant polynucleotide of  claim 44 , wherein the L chain variable region is fused to a gene activation moiety or a DNA-binding moiety.  
     
     
         46 . A recombinant polynucleotide comprising a coding sequence that encodes the H chain variable region of  claim 43 .  
     
     
         47 . The recombinant polynucleotide of  claim 46 , wherein the H chain variable region is fused to a gene activation moiety or a DNA-binding moiety.  
     
     
         48 . A vector comprising the recombinant polynucleotide of any one of claims  44 - 47 .  
     
     
         49 . The vector of  claim 48  being an expression vector.  
     
     
         50 . The vector of  claim 48  being a shuttle vector.  
     
     
         51 . A selectable library of expression vectors encoding a repertoire of L chain variable regions, each of said expression vectors comprising at least one recombinant polynucleotide sequence of  claim 44 .  
     
     
         52 . A selectable library of expression vectors encoding a repertoire of L chain variable regions, each of said expression vectors comprising at least one recombinant polynucleotide sequence of  claim 45 .  
     
     
         53 . The selectable library of  claim 51 , wherein at least one of said expression vectors is a shuttle vector.  
     
     
         54 . A selectable library of expression vectors encoding a repertoire of H chain variable regions, each of said expression vectors comprising at least one recombinant polynucleotide sequence of  claim 46 .  
     
     
         55 . A selectable library of expression vectors encoding a repertoire of H chain variable regions, each of said expression vectors comprising at least one recombinant polynucleotide sequence of  claim 47 .  
     
     
         56 . The selectable library of  claim 54 , wherein at least one of said expression vectors is a shuttle vector.  
     
     
         57 . A host cell expressing the recombinant polynucleotide of  claim 44  or  46 .  
     
     
         58 . The host cell of  57 , wherein the cell is eukaryotic.  
     
     
         59 . The host cell of  claim 58 , wherein the eukaryotic cell is a yeast cell.  
     
     
         60 . The host cell of  claim 57 , wherein the cell is prokaryotic.  
     
     
         61 . A method of screening for an antigen-binding component that is immunoreactive with a desired antigen, comprising: 
 (a) recombinantly co-expressing within a population of cells 
 (i) a first antigen fusion gene encoding the desired antigen fused in-frame with a first protein domain;  
 (ii) the selectable library of  claim 54 , wherein the H chain variable region is fused to a second protein domain;  
 (iii) a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first and second protein domains; and  
   (b) detecting expression of said reporter gene, wherein an increase in the expression indicates a specific binding between the antigen-binding component and the desired antigen, thereby identifying an antigen-binding component that is immunoreactive with the desired antigen.    
     
     
         62 . The method of  claim 61 , wherein the cells are yeast cells.  
     
     
         63 . The method of  claim 61 , wherein the first or second protein domain is a DNA-binding moiety.  
     
     
         64 . The method of  claim 61 , wherein the first or second protein domain is a gene activation moiety.  
     
     
         65 . The method of  claim 61 , further comprising the step of counter selecting cells that express the reporter gene independent of the specific interaction between the antigen-binding component and the desired antigen.  
     
     
         66 . The method of  claim 65 , wherein the step of counter selecting further comprising: 
 (a) recombinantly co-expressing within the population of cells 
 (i) a second antigen fusion gene encoding a second antigen fused in-frame with a third protein domain, wherein the second antigen differs structurally from the first antigen; and  
 (ii) a counterselectable gene wherein expression of the counterselectable gene is conditioned on the proximity of the second and third protein domains;  
   (b) culturing the cells under condition suitable for expression for the reporter gene and the counterselectable gene; and    (c) detecting expression of the reporter gene and the counterselectable gene, wherein an increase in the reporter gene expression in the absence of an increase in the counterselectable gene expression indicates that a specific binding between the antigen-binding component and the desired antigen has occurred.    
     
     
         67 . The method of  claim 61 , wherein the reporter gene is selected from the group consisting of LEU2, TRP1, HIS3, LacZ, URA3, and MEL.  
     
     
         68 . The method of  claim 63 , wherein said DNA-binding moiety comprises the DNA-binding domain of a protein selected from the group consisting of GAL4, LexA, and Ace1.  
     
     
         69 . The method of  claim 64 , wherein said gene activation moiety comprises the transcription activation domain selected from the group consisting of GAL4 and VP16.  
     
     
         70 . The method of  claim 66 , wherein said counterselectable gene is selected from the group consisting of URA3, LYS5, GAL1, CYH2, and CAN1.  
     
     
         71 . The method of  claim 70 , wherein said counterselectable gene is integrated into the genome of the population of cells, said cells being mating or mated yeast cells.  
     
     
         72 . The method of  claim 70 , wherein said expression of said counterselectable gene is lethal to the cell.  
     
     
         73 . The method of  claim 61 , wherein the antigen-binding component comprises a heavy chain variable region.  
     
     
         74 . A method of screening for an antigen-binding component that is immunoreactive with a desired antigen, comprising: 
 (a) recombinantly co-expressing within a population of cells 
 (i) a first antigen fusion gene encoding the desired antigen fused in-frame with a first protein domain;  
 (ii) the selectable library of  claim 51 , wherein the L chain variable region is fused to a second domain;  
 (iii) a reporter gene wherein expression of the reporter gene is conditioned on the proximity of the first and second protein domains; and  
   (b) detecting expression of said reporter gene, wherein an increase in the expression indicates a specific binding between the antigen-binding component and the desired antigen, thereby identifying an antigen-binding component that is immunoreactive with the desired antigen.    
     
     
         75 . The method of  claim 74 , wherein the cells are yeast cells.  
     
     
         76 . The method of  claim 74 , wherein the antigen-binding component comprises a light chain variable region.