Antibody libraries

Abstract

The present invention relates to the production of antibody libraries. In particular, the present invention relates to the use of integrating retroviral vectors to generate libraries comprising a plurality of combinations of antibody light chains and heavy chains. The present invention thus provides improved methods of generating and screening antibody libraries comprising large numbers of unique antibodies.

What is claimed is:  
     
         1 . An antibody library comprising at least 10 2  cells, wherein each cell comprises at least one integrated retroviral vector, wherein said retroviral vector expresses an antibody light chain.  
     
     
         2 . The antibody library of  claim 1 , wherein said library expresses at least 10 2  unique antibody light chains.  
     
     
         3 . The antibody library of  claim 1 , wherein said library expresses at least 10 3  unique antibody light chains.  
     
     
         4 . The antibody library of  claim 1 , wherein said library expresses at least 10 4  unique antibody light chains.  
     
     
         5 . The antibody library of  claim 1 , wherein said library expresses at least 10 5  unique antibody light chains.  
     
     
         6 . The antibody library of  claim 1 , wherein each of said cells comprises exactly one of said integrated retroviral vector.  
     
     
         7 . An antibody library comprising at least 10 2  cells, wherein each cell comprises at least one integrated retroviral vector, wherein said retroviral vector expresses an antibody heavy chain.  
     
     
         8 . The antibody library of  claim 7 , wherein said library expresses at least 10 2  unique antibody heavy chains.  
     
     
         9 . The antibody library of  claim 7 , wherein said library expresses at least 10 3  unique antibody heavy chains.  
     
     
         10 . The antibody library of  claim 7 , wherein said library expresses at least 10 4  unique antibody heavy chains.  
     
     
         11 . The antibody library of  claim 7 , wherein said library expresses at least 10 5  unique antibody heavy chains.  
     
     
         12 . The antibody library of  claim 7 , wherein each of said cells comprises exactly one of said integrated retroviral vector.  
     
     
         13 . An antibody library comprising at least 10 2  cells, wherein each cell comprises at least one of a first integrated retroviral vector and at least one of a second integrated retroviral vector, wherein said first retroviral vector expresses an antibody light chain and said second retroviral vector expresses an antibody heavy chain, and wherein said antibody light chain and said antibody heavy chain associate to form an antibody.  
     
     
         14 . The antibody library of  claim 13 , wherein said first and second integrated vectors are separately integrated.  
     
     
         15 . The antibody library of  claim 13 , wherein said library expresses at least 10 2  unique antibodies.  
     
     
         16 . The antibody library of  claim 13 , wherein said library expresses at least 10 3  unique antibodies.  
     
     
         17 . The antibody library of  claim 13 , wherein said library expresses at least 10 4  unique antibodies.  
     
     
         18 . The antibody library of  claim 13 , wherein said library expresses at least 10 5  unique antibodies.  
     
     
         19 . The antibody library of  claim 13 , wherein said cell comprises exactly one of said first integrated retroviral and exactly one of said second integrated retroviral vector.  
     
     
         20 . A retroviral particle library comprising at least 10 2  retroviral particles, wherein each retroviral particle comprises one antibody light chain gene.  
     
     
         21 . The retroviral particle library of  claim 20 , wherein said library comprises at least 10 2  unique antibody light chain genes.  
     
     
         22 . The retroviral particle library of  claim 20 , wherein said library comprises at least 10 3  unique antibody light chain genes.  
     
     
         23 . The retroviral particle library of  claim 20 , wherein said library comprises at least 10 4  unique antibody light chain genes.  
     
     
         24 . The retroviral particle library of  claim 20 , wherein said library comprises at least 10 5  unique antibody light chain genes.  
     
     
         25 . A retroviral particle library comprising at least 10 2  retroviral particles, wherein each retroviral particle comprises one antibody heavy chain gene.  
     
     
         26 . The retroviral particle library of  claim 25 , wherein said library comprises at least 10 2  unique antibody heavy chain genes.  
     
     
         27 . The retroviral particle library of  claim 25 , wherein said library comprises at least 10 unique antibody heavy chain genes.  
     
     
         28 . The retroviral particle library of  claim 25 , wherein said library comprises at least 10 4  unique antibody heavy chain genes.  
     
     
         29 . The retroviral particle library of  claim 25 , wherein said library comprises at least 10 5  unique antibody heavy chain genes.  
     
     
         30 . A retroviral particle library comprising at least 10 2  retroviral particles, wherein each retroviral particle comprises at least one antibody gene selected from the group consisting of antibody heavy chain genes and antibody light chain genes.  
     
     
         31 . The retroviral particle library of  claim 30 , wherein said library comprises at least 10 2  unique antibody genes.  
     
     
         32 . The retroviral particle library of  claim 30 , wherein said library comprises at least 10 3  unique antibody genes.  
     
     
         33 . The retroviral particle library of  claim 30 , wherein said library comprises at least 10 4  unique antibody genes.  
     
     
         34 . The retroviral particle library of  claim 30 , wherein said library comprises at least 10 5  unique antibody genes.  
     
     
         35 . The retroviral particle library of  claim 30 , wherein each retroviral particle comprises one antibody heavy chain gene and one antibody light chain gene.  
     
     
         36 . A plasmid library comprising at least 10 2  plasmids, wherein each plasmid comprises one antibody heavy chain gene inserted into a retroviral vector backbone.  
     
     
         37 . The plasmid library of  claim 36 , wherein said library comprises at least 10 2  unique antibody heavy chain genes.  
     
     
         38 . The plasmid library of  claim 36 , wherein said library comprises at least 10 3 unique antibody heavy chain genes.  
     
     
         39 . The plasmid library of  claim 36 , wherein said library comprises at least 10 4  unique antibody heavy chain genes.  
     
     
         40 . The plasmid library of  claim 36 , wherein said library comprises at least 10 5  unique antibody heavy chain genes.  
     
     
         41 . A plasmid library comprising at least 10 2  plasmids, wherein each plasmid comprises one antibody light chain gene inserted into a retroviral vector backbone.  
     
     
         42 . The plasmid library of  claim 41 , wherein said library comprises at least 10 2  unique antibody light chain genes.  
     
     
         43 . The plasmid library of  claim 41 , wherein said library comprises at least 10 3  unique antibody light chain genes.  
     
     
         44 . The plasmid library of  claim 41 , wherein said library comprises at least 10 4  unique antibody light chain genes.  
     
     
         45 . The plasmid library of  claim 41 , wherein said library comprises at least 10 5  unique antibody light chain genes.  
     
     
         46 . A plasmid library comprising at least 10 2  plasmids, wherein each plasmid comprises at least one antibody gene selected from the group consisting of antibody heavy chain gene and antibody light chain gene.  
     
     
         47 . The plasmid library of  claim 46 , wherein said library comprises at least 10 2  unique antibody genes.  
     
     
         48 . The plasmid library of  claim 46 , wherein said library comprises at least 10 3  unique antibody genes.  
     
     
         49 . The plasmid library of  claim 46 , wherein said library comprises at least 10 4  unique antibody genes.  
     
     
         50 . The plasmid library of  claim 46 , wherein said library comprises at least 10 5  unique antibody light chain genes.  
     
     
         51 . The plasmid library of  claim 46 , wherein each plasmid comprises one antibody heavy chain gene and one antibody light chain gene.  
     
     
         52 . A method of generating antibody libraries, comprising: 
 a) providing 
 i) a plurality of first integratable retroviral particles, wherein each of said plurality of retroviral particles comprises one antibody light chain gene;  
 ii) a plurality of second integratable retroviral particles, wherein each of said plurality of retroviral particles comprises one antibody heavy chain gene; and  
 iii) a host cell comprising a genome; and  
   b) contacting said plurality of host cell with said plurality of first and second integratable retroviral particles under conditions such that at least one of said plurality of first integratable retroviral particles and at least one of said plurality of second integratable retroviral particles integrate into said genome of said host cell to generate an antibody library.    
     
     
         53 . The method of  claim 52 , wherein said plurality of first integratable retroviral particles further comprises a first selectable marker, and said plurality of second integratable retroviral particles further comprises a second selectable marker.  
     
     
         54 . The method of  claim 53 , wherein said contacting further comprises selecting for the presence of said first and second selectable markers.  
     
     
         55 . The method of  claim 52 , wherein said antibody library comprises at least 10 2  unique antibodies.  
     
     
         56 . The method of  claim 52 , wherein said antibody library comprises at least 10 3  unique antibodies.  
     
     
         57 . The method of  claim 52 , wherein said antibody library comprises at least 10 4  unique antibodies.  
     
     
         58 . The method of  claim 52 , wherein said antibody library comprises at least 10 5  unique antibodies.  
     
     
         59 . The method of  claim 52 , wherein exactly one of said plurality of first integratable retroviral particles and exactly one of said plurality of second integratable retroviral particles integrate into said genome of said host cell.  
     
     
         60 . The method of  claim 52 , further comprising step c) screening said antibody library.  
     
     
         61 . The method of  claim 60 , wherein said screening comprises detecting the ability of antibodies in said antibody library to bind to a pre-selected antigen.  
     
     
         62 . The method of  claim 61 , wherein said antibodies are bound to the membrane of said host cell and said detecting comprises fluorescence activated cell sorting.  
     
     
         63 . The method of  claim 61 , wherein said antibodies are secreted and said detecting comprises a solution-based detection assay.  
     
     
         64 . The method of  claim 63 , wherein said antibodies are diluted into individual containers prior to said detecting.  
     
     
         65 . The method of  claim 63 , wherein said solution based assay is selected from the group consisting of radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays, immunoprecipitation reactions, agglutination assays (e.g., hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, and protein A assays.  
     
     
         66 . A method of screening antibody libraries, comprising: 
 a) providing 
 i) an antibody library comprising comprises at least 10 2  unique antibodies; and  
 ii) a pre-selected antigen; and  
   b) screening said antibody library, wherein said screening comprises detecting the ability of said at least 10 2  unique antibodies to bind to said pre-selected antigen.    
     
     
         67 . The method of  claim 66 , wherein said antibody library comprises at least 10 3  unique antibodies.  
     
     
         68 . The method of  claim 66 , wherein said antibody library comprises at least 10 4  unique antibodies.  
     
     
         69 . The method of  claim 66 , wherein said antibody library comprises at least 10 5  unique antibodies.  
     
     
         70 . The method of  claim 66 , wherein said antibodies are bound to the membrane of a host cell and said detecting comprises fluorescence activated cell sorting.  
     
     
         71 . The method of  claim 66 , wherein said antibodies are secreted and said detecting comprises a solution-based detection assay.  
     
     
         72 . The method of  claim 71 , wherein said antibodies are diluted into individual containers prior to said detecting.  
     
     
         73 . The method of  claim 71 , wherein said solution based assay is selected from the group consisting of radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays, immunoprecipitation reactions, agglutination assays (e.g., hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, and protein A assays.  
     
     
         74 . A method, comprising 
 a) providing 
 i) a plurality of first integratable retroviral particles, wherein each of said plurality of retroviral particles comprises one antibody light chain gene;  
 ii) a plurality of second integratable retroviral particles, wherein each of said plurality of retroviral particles comprises one antibody heavy chain gene; and  
 iii) a host cell comprising a genome; and  
 iv) a pre-selected antigen; and  
   b) contacting said plurality of host cell with said plurality of first and second integratable retroviral particles under conditions such that at least one of said plurality of first integratable retroviral particles and at least one of said plurality of second integratable retroviral particles integrate into said genome of said host cell to generate an antibody library comprising a plurality of antibodies; and    c) screening said antibody library, wherein said screening comprises detecting the ability said antibodies to bind to said pre-selected antigen.    
     
     
         75 . The method of  claim 74 , wherein said antibody library comprises at least 10 2  unique antibodies.  
     
     
         76 . The method of  claim 74 , wherein said antibody library comprises at least 10 3  unique antibodies.  
     
     
         77 . The method of  claim 74 , wherein said antibody library comprises at least 10 4  unique antibodies.  
     
     
         78 . The method of  claim 74 , wherein said antibody library comprises at least 10 5  unique antibodies.  
     
     
         79 . The method of  claim 74 , wherein said plurality of first integratable retroviral particles further comprises a first selectable marker, and said plurality of second integratable retroviral particles further comprises a second selectable marker.  
     
     
         80 . The method of  claim 79 , wherein said contacting further comprises selecting for the presence of said first and second selectable markers.  
     
     
         81 . The method of  claim 74 , wherein said antibodies are bound to the membrane of said host cell and said detecting comprises fluorescence activated cell sorting.  
     
     
         82 . The method of  claim 74 , wherein said antibodies are secreted and said detecting comprises a solution-based detection assay.  
     
     
         83 . The method of  claim 82 , wherein said antibodies are diluted into individual containers prior to said detecting.  
     
     
         84 . The method of  claim 82 , wherein said solution based assay is selected from the group consisting of radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays, immunoprecipitation reactions, agglutination assays (e.g., hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, and protein A assays.