Protein combination-based fv library, and preparation method therefor

Abstract

The present invention relates to a method for constructing an Fv library based on a combination of proteins, a method of screening a desired antibody using the constructed Fv library, an Fv antibody screened by the screening method, and an Fv library constructed by the Fv library construction method. The Fv library of the present invention is based on a combination of proteins so that members thereof can be individually analyzed for their function. Moreover, the Fv library enables a desired Fv antibody to be screened without needing a target antigen preparation. In addition, the protein combination based Fv library makes it possible to significantly reduce the number of protein purification processes to thereby reduce costs and time, compared to conventional DNA-based libraries.

1 - 21 . (canceled) 
     
     
         22 . A method for constructing an Fv library comprising:
 (a) expressing and purifying individual heavy-chain variable region (VH) domain proteins from cells;   (b) expressing and purifying light-chain variable region (VL) domain proteins from cells separate from the cells in (a); and   (c) pairing the purified VH domain proteins and the purified VL domain proteins to one another.   
     
     
         23 . A method for screening a desired VH domain protein and VL domain protein pair, the method comprising the steps of:
 (a) performing functional analysis on individual members of a Fv library constructed according to the method of  claim 22 ; and   (b) identifying a VH domain protein and VL domain protein pair that exhibits a desired property, characteristic, or activity.   
     
     
         24 . An Fv library comprising:
 (a) individual heavy-chain variable region (VH) domain proteins expressed and purified from cells;   (b) individual light-chain variable region (VL) domain proteins expressed and purified from cells separate from the cells in (a); and   wherein the purified VH domain proteins and the purified VL domain proteins are paired to one another.   
     
     
         25 . The Fv library of  claim 24 , wherein the VH domain proteins comprise amino acid sequences that differ from each other. 
     
     
         26 . The Fv library of  claim 24 , wherein the VL domain proteins comprise amino acid sequences that differ from each other. 
     
     
         27 . The Fv library of  claim 24 , wherein VH domain proteins comprise amino acid sequences that differ from each other and the VL domain proteins comprise amino acid sequences that differ from each other. 
     
     
         28 . The Fv library of  claim 24 , wherein the VH domain proteins derive from the same original VH domain and the VH domain proteins comprise amino acid sequences that differ from each other; or wherein the VL domain proteins derive from the same original VL domain and the VL domain proteins comprise amino acid sequences that differ from each other. 
     
     
         29 . The Fv library of  claim 24 , wherein the VH domain proteins derive from the same original VH domain and the VH domain proteins comprise amino acid sequences that differ from each other; and wherein the VL domain proteins derive from the same original VL domain and the VL domain proteins comprise amino acid sequences that differ from each other. 
     
     
         30 . The Fv library of  claim 24 , wherein the VH domain proteins comprise complementarity determining regions (CDRs) and framework regions (FRs) and the VL domain proteins comprise complementarity determining regions (CDRs) and framework regions (FRs). 
     
     
         31 . The Fv library of  claim 30 , wherein CDRs of the VH domain proteins comprise amino acid sequences that differ from each other; CDRs of the VL domain proteins comprise amino acid sequences that differ from each other; FRs of the VH domain proteins comprise amino acid sequences that differ from each other; or FRs of the VL domain proteins comprise amino acid sequences that differ from each other. 
     
     
         32 . The Fv library of  claim 31 , wherein CDRs of the VH domain proteins comprise amino acid sequences that differ from each other; and CDRs of the VL domain proteins comprise amino acid sequences that differ from each other. 
     
     
         33 . The Fv library of  claim 31 , wherein FRs of the VH domain proteins comprise amino acid sequences that differ from each other; and FRs of the VL domain proteins comprise amino acid sequences that differ from each other. 
     
     
         34 . The Fv library of  claim 32 , wherein FRs of the VH domain proteins comprise amino acid sequences that differ from each other; and FRs of the VL domain proteins comprise amino acid sequences that differ from each other. 
     
     
         35 . The Fv library of  claim 24 , wherein the VH domain proteins and the VL domain proteins are paired by random pairing or target pairing. 
     
     
         36 . The Fv library of  claim 24 , wherein the pairing of the VH domain proteins with the VL domain proteins is performed by a method comprising: (i) pairing between wild-type VH domain proteins and the VL domain proteins; (ii) pairing by disulfide bonds between cysteine residues introduced into each of the VH domain proteins and the VL domain proteins; (iii) pairing by fusion between coiled-coil domains introduced into each of the VH domain proteins and the VL domain proteins; or (iv) pairing by protein-protein interaction between proteins fused to each of the VH domain proteins and the VL domain proteins. 
     
     
         37 . The Fv library of  claim 24 , wherein the paired VH domain proteins and the VL domain proteins are stored in individual compartments. 
     
     
         38 . The Fv library of  claim 37 , wherein the individual compartments are assigned a unique identification (ID) number. 
     
     
         39 . The Fv library of  claim 37 , wherein the compartments comprise plate wells, test tubes, microfluidic channels, or chips. 
     
     
         40 . The Fv library of  claim 24 , wherein the Fv library enables functional analysis of individual members thereof. 
     
     
         41 . The Fv library of  claim 40 , wherein the functional analysis does not comprise a pre-screening step based on binding to a target. 
     
     
         42 . The Fv library of  claim 40 , wherein the desired property, characteristic, or activity is cell proliferation, differentiation, or cell death.