Method to screen phage display libraries with different ligands

Abstract

Abstract of the Disclosure The present invention relates to methods for selecting repertoires of polypeptides using generic and target ligands. In particular, the invention relates to a library comprising a repertoire of polypeptides of the immunoglobulin superfamily, wherein the members of the repertoire have a known main chain conformation.

What is Claimed is: 
     
         1.  150.  A synthetic library of antibody polypeptides wherein each member of said library comprises a V H  domain polypeptide sequence that comprises a V H  hypervariable loop with the canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47. 
     
     
         2.  [c3] 
         151.   A library comprising 2 x 10 8  or more antibody polypeptides comprising V H  domains, wherein each said V H  domain comprises an H loop with the canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47. 
     
     
         3.  [c4] 
         152.   The library of claim  150  or  151  wherein said hypervariable loop is loop H1. 
     
     
         4.  [c5] 
         153.   The library of claim  150  or  151  wherein said hypervariable loop is H2. 
     
     
         5.  [c6] 
         154.   The library of claim  150  or  151  wherein the members of said library comprise hypervariable loops that have the canonical structures of hypervariable loops H1 and H2 encoded by human germline V H  gene segment DP-47. 
     
     
         6.  [c7] 
         155.   A synthetic library of antibody polypeptides wherein the members of said library comprise a V H  domain polypeptide sequence having the framework regions encoded by human germline V H  gene segment DP-47. 
     
     
         7.  156.  The synthetic library of claim  155  wherein the members of said library further comprise one or more hypervariable loops that have the canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47. 
     
     
         8.  [c8] 
         157.   The synthetic library of claim  156  wherein the members of said library comprise hypervariable loops that have the canonical structures of hypervariable loops H1 and H2 encoded by human germline V H  gene segment DP-47. 
     
     
         9.  [c9] 
         158.   The synthetic library of claim  156  wherein said one or more hypervariable loops is diversified at one or more residues through use of an NNK codon, a DVT codon or a DVY codon. 
     
     
         10.  [c10] 
         159.   The library of claim  150  or  151  wherein said hypervariable loop has the canonical structure of loop H1 encoded by human germline V H  gene segment DP-47, and wherein said loop is diversified at one or more residues through use of an NNK codon, a DVT codon or a DVY codon. 
     
     
         11.  [c11] 
         160.   The library of claim  150  or  151  wherein said hypervariable loop has the canonical structure of loop H1 encoded by human germline V H  gene segment DP-47, and wherein said loop is diversified at one or more residues selected from the group consisting of H31, H33 and H35. 
     
     
         12.  [c12] 
         161.   The library of claim  160  wherein said loop is diversified through use of an NNK codon, a DVT codon or a DVY codon. 
     
     
         13.  [c13] 
         162.   The library of claim  150  or  151  wherein said hypervariable loop has the canonical structure of loop H2 encoded by human germline V H  gene segment DP-47, and wherein said loop is diversified at one or more residues through use of an NNK codon, a DVT codon or a DVY codon. 
     
     
         14.  [c14] 
         163.   The library of claim  150  or  151  wherein said hypervariable loop has the canonical structure of loop H2 encoded by human germline V H  gene segment DP-47, and wherein said loop is diversified at one or more residues selected from the group consisting of H50, H52, H52a, H53, H55, H56 and H58. 
     
     
         15.  [c15] 
         164.   The library of claim  163  wherein said loop is diversified through use of an NNK codon, a DVT codon or a DVY codon. 
     
     
         16.  [c16] 
         165.   The library of claim  154  wherein said hypervariable loops that have the canonical structures of hypervariable loops H1 and H2 encoded by human germline V H  gene segment DP-47 are diversified at one or more residues through use of an NNK codon, a DVT codon or a DVY codon. 
     
     
         17.  [c17] 
         166.   The library of claim  165  wherein said hypervariable loops are diversified at one or more residues selected from the group consisting of H31, H33, H35, H50, H52, H52a, H53, H55 and H56. 
     
     
         18.  [c18] 
         167.   The library of claim  165  wherein the members of said library are diversified at each of residues H31, H33, H35, H50, H52, H52a, H53, H55 and H56. 
     
     
         19.  [c19] 
         168.   The library of claim  150  or  151  wherein said antibody polypeptides comprise the sequence of amino acids 1-116 of SEQ ID NO: 2, wherein a residue selected from the group consisting of H31, H33, H35, H50, H52, H52a, H53, H55 and H56 differs from the residue at that position in SEQ ID NO: 2. 
     
     
         20.  [c20] 
         169.   The library of claim  150  or  151  wherein said antibody polypeptides further comprise a V L  polypeptide sequence. 
     
     
         21.  [c21] 
         170.   The library of claim  169  wherein said antibody polypeptides are scFv or Fab polypeptides. 
     
     
         22.  [c22] 
         171.   The library of claim  150  or  151  wherein the members of said library bind the generic ligand Protein A. 
     
     
         23.  [c23] 
         172.   A method of making a library of antibody polypeptides, the method comprising: 
       a) providing a plurality of nucleic acids consisting of nucleic acids each encoding an antibody polypeptide comprising a V H  domain, wherein each V H  domain comprises H1 and H2 hypervariable loops encoded by human germline V H  gene segment DP-47, 
       b) introducing diversity into nucleic acids comprised by said plurality to provide diversity at one or more amino acid residues within one or more CDRs of a plurality of said V H  domains, ; and 
       c) expressing the polypeptides encoded by said plurality of nucleic acids, whereby a library of antibody polypeptides comprising diversified V H  domains is produced. 
     
     
         24.  [c24] 
         173.   The method of claim  172 , wherein in step (a), said plurality of nucleic acids are identical. 
     
     
         25.  [c25] 
         174.   The method of claim  172 , wherein in step (a), said plurality of nucleic acids encode identical V H  domains. 
     
     
         26.  [c26] 
         175.   The method of claim  172  wherein said diversity is introduced through the use of an NNK codon, a DVT codon or a DVY codon. 
     
     
         27.  [c27] 
         176.   The method of claim  172  wherein said diversity is introduced at selected positions within one or both of hypervariable loops H1 and H2 of said plurality of V H  domains. 
     
     
         28.  [c28] 
         177.   The method of claim  172  wherein said diversity is introduced at one or more of amino acid residues selected from the group consisting of H31, H33, H35, H50, H52, H52a, H53, H55, H56, H95, H96, H97 and H98 encoded by human germline V H  gene segment DP-47. 
     
     
         29.  [c29] 
         178.   The method of claim  177  wherein said diversity is introduced at each of the amino acid residues H31, H33, H35, H50, H52, H52a, H53, H55, H56, H95, H96, H97 and H98 encoded by human germline V H  gene segment DP-47.