Method to screen phage display libraries with different ligands

Abstract

The invention provides a method for selecting, from a repertoire of polypeptides, a population of functional polypeptides which bind a target ligand in a first binding site and a generic ligand in a second binding site, which generic ligand is capable of binding functional members of the repertoire regardless of target ligand specificity, comprising the steps of: a) contacting the repertoire with the generic ligand and selecting functional polypeptides bound thereto; and b) contacting the selected functional polypeptides with the target ligand and selecting a population of polypeptides which bind to the target ligand. The invention accordingly provides a method by which a polypeptide repertoire is preselected, according to functionality as determined by the ability to bind the generic ligand, and the subset of polypeptides obtained as a result of such preselection is then employed for further selection according to the ability to bind the target ligand.

1 . A naïve library of antibody polypeptides wherein each member of said library comprises a V H  domain polypeptide sequence having a framework region encoded by human germline V H  gene segment DP-47.  
     
     
         2 . The naïve library of  claim 1  wherein said framework region encoded by human germline VH gene segment DP-47 is FW1.  
     
     
         3 . The naïve library of  claim 1  wherein said framework region encoded by human germline VH gene segment DP-47 is FW2.  
     
     
         4 . The naïve library of  claim 1  wherein said framework region encoded by human germline VH gene segment DP-47 is FW3.  
     
     
         5 . The naive library of  claim 1  wherein said VH domain comprises a hypervariable loop diversified at one or more residues selected from the group consisting of H31, H33 and H35.  
     
     
         6 . The naïve library of  claim 5  wherein said hypervariable loop is diversified at each of residues H31, H33 and H35.  
     
     
         7 . A naïve library of antibody polypeptide members wherein each said member comprises a V H  loop generated by a process comprising in vitro diversification of human germline V H  gene segment DP-47.  
     
     
         8 . A naïve library of antibody polypeptides wherein each member of said library comprises a human V H  domain polypeptide sequence having one or more hypervariable loops that have the canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47.  
     
     
         9 . The library of  claim 8  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.  
     
     
         10 . The library of  claim 8  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.  
     
     
         11 . A naïve library of antibody polypeptides wherein each member of said library comprises a V H  domain polypeptide comprising a hypervariable loop that has the canonical structure of loop H1 encoded by human germline VH gene segment DP-47, and wherein the hypervariable loop has been diversified at one or more residues selected from the group consisting of H31, H33 and H35.  
     
     
         12 . The naïve library of  claim 8  wherein said VH is a human VH.  
     
     
         13 . The naïve library of  claim 8  wherein said hypervariable loop has been diversified at each of residues H31, H33 and H35.  
     
     
         14 . The library of  claim 1  wherein the members of said library further comprise a hypervariable loop that has the canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47, wherein said loop was generated by a process comprising in vitro diversification of human germline V H  gene segment DP-47.  
     
     
         15 . The library of  claim 13  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.  
     
     
         16 . A naïve library comprising 2×10 8  or more antibody polypeptides comprising V H  domains, wherein each said V H  domain comprises an H loop with canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47.  
     
     
         17 . A naïve library comprising 2×10 8  or more antibody polypeptides wherein each said antibody polypeptide comprises a V H  domain that comprises an H loop with canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47, and wherein said H loop comprises a sequence generated by a process comprising in vitro diversification of human germline V H  gene segment DP-47.  
     
     
         18 . The library of  claim 8  or  16  wherein said hypervariable loop is loop H1.  
     
     
         19 . The library of  claim 8  or  16  wherein said hypervariable loop is H2.  
     
     
         20 . The library of  claim 8  or  16  wherein members of said library comprise hypervariable loops that have canonical structures of hypervariable loops H1 and H2 encoded by human germline V H  gene segment DP-47.  
     
     
         21 . The library of  claim 8  or  16  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.  
     
     
         22 . The library of  claim 8  or  16  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.  
     
     
         23 . The library of  claim 22  wherein said loop is diversified through use of an NNK codon, a DVT codon or a DVY codon.  
     
     
         24 . The library of  claim 8  or  16  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.  
     
     
         25 . The library of  claim 8  or  16  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.  
     
     
         26 . The library of  claim 25  wherein said loop is diversified through use of an NNK codon, a DVT codon or a DVY codon.  
     
     
         27 . The library of  claim 20  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.  
     
     
         28 . The library of  claim 27  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.  
     
     
         29 . The library of  claim 27  wherein the members of said library are diversified at each of residues H31, H33, H35, H50, H52, H52a, H53, H55 and H56.  
     
     
         30 . The library of  claim 8  or  16  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.  
     
     
         31 . The library of  claim 8  or  16  wherein said antibody polypeptides further comprise a V L  polypeptide sequence.  
     
     
         32 . The library of  claim 31  wherein said antibody polypeptides are scFv or Fab polypeptides.  
     
     
         33 . The library of  claim 8  or  16  wherein the members of said library bind the generic ligand Protein A.  
     
     
         34 . A naïve library of antibody polypeptides wherein each member of said library comprises a V H  domain polypeptide sequence having: 
 a) framework regions 1 and 2 encoded by human germline VH gene segment DP-47; and    b) H1 and H2 hypervariable loops that have the canonical structures of H1 and H2 hypervariable loops encoded by human germline VH gene segment DP-47, wherein the sequence of hypervariable loop H1 in members of said library has been diversified at each of positions H31, H33 and H35.    
     
     
         35 . A naïve 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 canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47, and wherein each member of said library comprises an isoleucine at position 51 wherein residues are numbered according to the Kabat numbering convention.  
     
     
         36 . The library of  claim 35  wherein each member of said library comprises glycine at position 44.  
     
     
         37 . The library of  claim 35  wherein each member of said library comprises leucine at position 45.  
     
     
         38 . The library of  claim 35  wherein each member of said library comprises tryptophan at position 47.  
     
     
         39 . The library of  claim 35  wherein each member of said library comprises glycine at position 44 and leucine at position 45.  
     
     
         40 . The library of  claim 35  wherein each member of said library comprises glycine at position 44 and tryptophan at position 47.  
     
     
         41 . The library of  claim 35  wherein each member of said library comprises leucine at position 45 and tryptophan at position 47.  
     
     
         42 . The library of  claim 35  wherein each member of said library comprises glycine at position 44, leucine at position 45 and tryptophan at position 47.  
     
     
         43 . The library of  claim 35  wherein each member of said library comprises methionine at position 34.  
     
     
         44 . A naïve library comprising 2×10 8  or more antibody polypeptides comprising V H  domains, wherein each said V H  domain comprises an H loop with canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47, wherein each member of said library comprises an isoleucine at position 51 wherein residues are numbered according to the Kabat numbering convention.  
     
     
         45 . The library of  claim 44  wherein each member of said library comprises glycine at position 44.  
     
     
         46 . The library of  claim 44  wherein each member of said library comprises leucine at position 45.  
     
     
         47 . The library of  claim 44  wherein each member of said library comprises tryptophan at position 47.  
     
     
         48 . The library of  claim 44  wherein each member of said library comprises glycine at position 44 and leucine at position 45.  
     
     
         49 . The library of  claim 44  wherein each member of said library comprises glycine at position 44 and tryptophan at position 47.  
     
     
         50 . The library of  claim 44  wherein each member of said library comprises leucine at position 45 and tryptophan at position 47.  
     
     
         51 . The library of  claim 44  wherein each member of said library comprises glycine at position 44, leucine at position 45 and tryptophan at position 47.  
     
     
         52 . The library of  claim 44  wherein each member of said library comprises methionine at position 34.  
     
     
         53 . A naïve library of antibody polypeptides wherein each member of said library comprises a V H  domain polypeptide sequence having a framework region encoded by human germline V H  gene segment DP-47, wherein each member of said library comprises an isoleucine at position 51 wherein residues are numbered according to the Kabat numbering convention.  
     
     
         54 . The naïve library of  claim 53  wherein said framework region encoded by human germline V H  gene segment DP-47 is FW1.  
     
     
         55 . The naïve library of  claim 53  wherein said framework region encoded by human germline V H  gene segment DP-47 is FW2.  
     
     
         56 . The naïve library of  claim 53  wherein said framework region encoded by human germline V H  gene segment DP-47 is FW3.  
     
     
         57 . The library of  claim 53  wherein each member of said library comprises glycine at position 44.  
     
     
         58 . The library of  claim 53  wherein each member of said library comprises leucine at position 45.  
     
     
         59 . The library of  claim 53  wherein each member of said library comprises tryptophan at position 47.  
     
     
         60 . The library of  claim 53  wherein each member of said library comprises glycine at position 44 and leucine at position 45.  
     
     
         61 . The library of  claim 53  wherein each member of said library comprises glycine at position 44 and tryptophan at position 47.  
     
     
         62 . The library of  claim 53  wherein each member of said library comprises leucine at position 45 and tryptophan at position 47.  
     
     
         63 . The library of  claim 53  wherein each member of said library comprises glycine at position 44, leucine at position 45 and tryptophan at position 47.  
     
     
         64 . The library of  claim 53  wherein each member of said library comprises methionine at position 34.