Methods for producing members of specific binding pairs

Abstract

A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

1 . A phagemid comprising a DNA encoding a single-chain antibody-coliphage pill fusion protein, wherein the fusion protein contains a contiguous full length coliphage pIII protein. 
     
     
         2 . The phagemid according to  claim 1 , wherein the fusion protein contains a protease-sensitive region between the antibody and the coliphage pIII protein. 
     
     
         3 . The phagemid according to either  claim 1  or  claim 2 , wherein the phagemid comprises the genetic elements and DNA sequences (SEQ ID NOS: 7-17) of  FIGS. 1(   a )- 1 ( c ). 
     
     
         4 . A process of producing the phagemid according to  claim 1 , comprising fusing a DNA encoding the single-chain antibody to a DNA encoding the contiguous full length coliphage pIII protein and inserting the resulting DNA into a phagemid vector. 
     
     
         5 . The process according to  claim 4 , further comprising inserting a DNA encoding a protease-sensitive region between the DNA encoding the single-chain antibody and the DNA encoding the coliphage pIII protein. 
     
     
         6 . A method of selecting antibodies from an antibody library, comprising screening said antibody library with an antigen, wherein said antibody library consists of phagemids according to either  claim 1  or  claim 2 . 
     
     
         7 . A method of presenting a peptide or protein at the surface of a phagemid viral particle, comprising producing said phagemid viral particle, wherein said phagemid viral particle comprises a DNA sequence encoding said peptide or protein fused to a DNA sequence encoding a contiguous full length coliphage pIII protein. 
     
     
         8 . A filamentous bacteriophage particle displaying on its surface as a fusion with a gene III coat protein surface component a member of a specific binding pair in functional form comprising a binding domain for a complementary specific binding pair member, the particle containing a phagemid genome which is plasmid nucleic acid containing a single stranded phage replication origin and a nucleotide sequence encoding said fusion, and the particle having a coat partially derived from a helper phage and partly from said fusion. 
     
     
         9 . A particle according to  claim 8  wherein the displayed specific binding pair member comprises a binding domain of an immunoglobulin. 
     
     
         10 . A particle according to  claim 9  wherein the specific binding pair member is a scFv molecule. 
     
     
         11 . A phagemid comprising DNA encoding a polypeptide-coliphage pIII fusion protein, wherein said fusion protein comprises a single-chain polypeptide and a functional coliphage pIII polypeptide, and said functional coliphage pIII polypeptide comprises contiguous amino and carboxy domains of a coliphage pIII protein. 
     
     
         12 . A phagemid according to  claim 11 , wherein the single-chain polypeptide is a single-chain antibody. 
     
     
         13 . A phagemid according to  claim 11 , wherein said polypeptide-coliphage pIII fusion protein contains a protease-sensitive site between the single-chain polypeptide and the coliphage pIII polypeptide. 
     
     
         14 . A phagemid according to  claim 11  or  claim 12 , further comprising expression control elements upstream of said DNA and further encoding at least one selectable marker. 
     
     
         15 . A process for the production of a phagemid according to  claim 11 , comprising fusing a DNA encoding a single-chain polypeptide to a DNA encoding a functional coliphage pill polypeptide, wherein said functional coliphage pill polypeptide comprises contiguous amino and carboxy domains of a coliphage pIII protein, and inserting the resulting DNA molecule into a phagemid. 
     
     
         16 . The process according to  claim 15 , further comprising inserting a protease-sensitive site between the DNA encoding the single-chain polypeptide and the DNA encoding the coliphage pIII polypeptide. 
     
     
         17 . A phagemid according to  claim 11 , wherein said coliphage pIII polypeptide is a full-length coliphage pIII protein. 
     
     
         18 . A method of screening for binding ligands, comprising exposing ligands to a single chain polypeptide-coliphage pIII protein expressed by the phagemid of  claim 11  and selecting those ligands which recognize and bind to the single chain polypeptide-coliphage pIII protein.