Novel Method for Cloning Variable Domain Sequences of Immunological Gene Repertoire

Abstract

The present invention relates to a non-PCR (polymerase chain reaction) process, particularly a transcription-based amplification method, for amplifying and cloning sequences containing a variable domain sequence such as an immunoglobulin variable domain sequence from the immunological gene repertoire. The present invention contemplates the expression of antibody library in either in an in vivo expression vector or in an in vitro transcription/translation system. Isolation of a gene coding for a receptor having the ability to bind a preselected ligand and receptors produced by the gene isolated by the method is also contemplated.

1 . A process for isolating a nucleic acid encoding a protein that binds to a target antigen, comprising:
 (a) exposing a plurality of nucleic acids encoding a plurality of proteins that bind a plurality of antigens to at least one primer under conditions suitable to produce a DNA comprising a nucleic acid that functions as an RNA polymerise promoter;   (b) transcribing said DNA of step (a) with a suitable RNA polymerase to produce plural RNAs;   (c) optionally, repeating steps (a) and (b) to produce plural copies of said DNA or said RNA;   (d) cloning said DNA, RNA or functional parts thereof under conditions that enable expression of said DNA;   (e) expressing proteins encoded by said cloned DNA or RNA of step (d); and   (f) screening said expressed proteins with said target antigen to identify a protein that specifically binds thereto, thereby identifying a nucleic acid encoding a protein that binds to said antigen;   wherein when one primer is used, said primer comprises said RNA polymerase promoter, and when two primers are used, either primer comprises said promoter.   
     
     
         2 . The process of  claim 1 , wherein said nucleic acid is an RNA. 
     
     
         3 . The process of  claim 1 , wherein said protein comprises V H , V L  or a combination thereof. 
     
     
         4 . The process of  claim 3 , wherein said protein comprises V H  and V L . 
     
     
         5 . The process of  claim 4 , wherein said protein further comprises a linker molecule between said V H  and said V L . 
     
     
         6 . The process of  claim 1  wherein said conditions that enable expression comprise a vector in a cell, an in vitro transcription/translation reaction mixture or a combination of both. 
     
     
         7 . The process of  claim 1 , wherein a first primer is used to produce a complement, wherein said complement is treated to yield a 3′ poly C tail and a second primer comprises poly G and said promoter. 
     
     
         8 . The process of  claim 1  further comprising modifying said RNA, said DNA or both to yield additional proteins that bind antigen. 
     
     
         9 . The process of  claim 8 , wherein said modifying is by mutagenesis. 
     
     
         10 . A library of proteins, nucleic acids, cells comprising said nucleic acids or cells expressing said proteins made by the method of  claim 1 . 
     
     
         11 . The library of  claim 10 , comprising at least 10 4  proteins. 
     
     
         12 . The library of  claim 11 , comprising at least 10 5  proteins. 
     
     
         13 . The library of  claim 12 , comprising at least 10 6  proteins. 
     
     
         14 . A process for isolating a nucleic acid encoding a protein that binds to a target antigen, comprising:
 (a) exposing a plurality of nucleic acids encoding different proteins that bind to different antigens to non-PCR amplification to yield amplified nucleic acid sequences;   (b) cloning said amplified nucleic acid sequences or functional parts thereof in an expression system;   (c) expressing proteins encoded by the cloned nucleic acid sequences to form a library of expressed proteins capable of binding different antigens; and   (d) identifying a protein that binds to said target antigen, thereby identifying the nucleic acid encoding said protein that binds to said target antigen.   
     
     
         15 . The process of  claim 14 , wherein said amplification comprises RNA transcription. 
     
     
         16 . The process of  claim 14 , wherein said amplification comprises strand displacement. 
     
     
         17 . The process of  claim 14 , wherein said amplification comprises an RNA replicase activity. 
     
     
         18 . The process of  claim 14 , wherein said amplification comprises rolling circle amplification. 
     
     
         19 . The process of  claim 14 , wherein the protein is selected from the group consisting of V H , V L , and combinations thereof. 
     
     
         20 . The process of  claim 19 , wherein the protein comprises a V H  and a V L  and further comprises a linker bonded between the V H  and the V L . 
     
     
         21 . The process of  claim 14 , wherein said expression system is selected from the group consisting of an expression system comprising a vector and a host cell, an in vitro transcription and translation expression system or a combination thereof; and wherein said proteins are expressed using a vector and a host cell, an in vitro transcription and translation expression system or a combination thereof. 
     
     
         22 . The process of  claim 14 , further comprising modifying said amplified nucleic acid to yield additional proteins that bind antigens. 
     
     
         23 . The process of  claim 22 , wherein said modifying is by mutagenesis. 
     
     
         24 . A library of proteins or nucleic acid sequences encoding said proteins made by the process of  claim 14 . 
     
     
         25 . The library of  claim 24 , encoding at least 10 4  proteins. 
     
     
         26 . The library of  claim 25 , encoding at least 10 5  proteins. 
     
     
         27 . The library of  claim 26 , encoding at least 10 6  proteins. 
     
     
         28 . A process for making a library of proteins that bind to antigens, comprising:
 (a) exposing a plurality of nucleic acids encoding different proteins that bind different antigens to at least one primer under conditions suitable to produce a DNA comprising a nucleic acid that functions as an RNA polymerase promoter;   (b) transcribing the DNA sequence of step (a) with a suitable RNA polymerase to produce plural RNAS;   (c) optionally, repeating steps (a) and (b) to produce plural copies of said DNA sequence or said RNA sequence;   (d) cloning said DNA sequence or said RNA sequence or functional parts thereof in an expression system; and   (e) expressing proteins encoded by the cloned DNA sequence or RNA sequence or fragment thereof to form a library of expressed proteins capable of binding different antigens.   
     
     
         29 . The process of  claim 28 , further comprising screening the expressed proteins with an antigen to identify a protein that binds to said antigen. 
     
     
         30 . The process of  claim 28 , wherein said primer comprises said nucleic acid that functions as an RNA polymerase promoter when one primer is used and when two primers are used, either primer comprises the promoter. 
     
     
         31 . The process of  claim 28 , wherein said nucleic acids are RNA. 
     
     
         32 . The process of  claim 28 , wherein said proteins are selected from the group consisting of V H , V L , and combinations thereof. 
     
     
         33 . The process of  claim 32 , wherein said proteins further comprise a linker bonded between the V H  and the V L . 
     
     
         34 . The process of  claim 28 , wherein said expression system is selected from a group consisting of an expression system comprising a vector and a host cell, an in vitro transcription and translation expression system or a combination thereof; and wherein said proteins are expressed using a vector and a host cell, an in vitro transcription and translation expression system, or a combination thereof. 
     
     
         35 . The process of  claim 28 , wherein a first primer is used to produce a complement, wherein said complement is treated to yield a 3′ poly C tail and a second primer comprises poly G and said promoter sequence. 
     
     
         36 . The process of  claim 28 , further comprising modifying said RNA, said DNA or both to yield additional proteins that bind antigens. 
     
     
         37 . The process of  claim 36 , wherein said modifying is by mutagenesis. 
     
     
         38 . A library of proteins made by the process of  claim 28 . 
     
     
         39 . The library of  claim 38 , encoding at least 10 4  proteins. 
     
     
         40 . The library of  claim 39 , encoding at least 10 5  proteins. 
     
     
         41 . The library of  claim 40 , encoding at least 10 6  proteins.