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 . An antibody polypeptide comprising a specific binding site for a human polypeptide antigen, the antibody polypeptide comprising a heavy chain variable domain that provides the binding site for said human polypeptide antigen and comprises a hypervariable loop that has the canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47, wherein said variable domain is a selected member of a repertoire of antibody polypeptides, and wherein each member of said repertoire comprises a single variable domain.  
     
     
         2 . The antibody polypeptide of  claim 1  wherein said hypervariable loop has the canonical structure of a hypervariable loop H1 encoded by human germline V H  gene segment DP-47.  
     
     
         3 . The antibody polypeptide of  claim 1  wherein said hypervariable loop has the canonical structure of a hypervariable loop H2 encoded by human germline V H  gene segment DP-47.  
     
     
         4 . The antibody polypeptide of  claim 1  which is a Fab or F(ab′)2 polypeptide.  
     
     
         5 . The antibody polypeptide of  claim 1  which is an scFv polypeptide.  
     
     
         6 . The antibody polypeptide of  claim 1  wherein said repertoire is a synthetic repertoire.  
     
     
         7 . The antibody polypeptide of  claim 1  wherein said repertoire is an immune repertoire.  
     
     
         8 . The antibody polypeptide of  claim 1  wherein the amino acid corresponding to position H44 according to the Kabat numbering convention is glycine.  
     
     
         9 . The antibody polypeptide of  claim 1  wherein the amino acid corresponding to position H45 according to the Kabat numbering convention is leucine.  
     
     
         10 . The antibody polypeptide of  claim 1  wherein the amino acid corresponding to position H47 according to the Kabat numbering convention is tryptophan.  
     
     
         11 . The antibody polypeptide of  claim 1  wherein the amino acids corresponding to amino acid positions H44, H45 and H47 according to the Kabat numbering convention are glycine, leucine and tryptophan, respectively.  
     
     
         12 . The antibody polypeptide of  claim 1  wherein members of said repertoire vary at selected positions.  
     
     
         13 . The antibody polypeptide of  claim 1  wherein the CDR1 amino acid sequence of said heavy chain variable domain varies from the heavy chain variable domain CDR1 sequence of  FIG. 2  at position H31, H33 and/or H35.  
     
     
         14 . The antibody polypeptide of  claim 1  wherein the CDR1 amino acid sequence of said heavy chain variable domain varies from the heavy chain variable domain CDR1 sequence of  FIG. 2  only at position H31, H33 and/or H35.  
     
     
         15 . The antibody polypeptide of  claim 1  wherein the CDR2 amino acid sequence of said heavy chain variable domain varies from the heavy chain variable domain CDR2 sequence of  FIG. 2  at position H50, H52, H52a, H53, H55, H56 and/or H58.  
     
     
         16 . The antibody polypeptide of  claim 1  wherein the CDR2 amino acid sequence of said heavy chain variable domain varies from the heavy chain variable domain CDR2 sequence of  FIG. 2  only at position H50, H52, H52A, H53, H55, H56 and/or H58.  
     
     
         17 . The antibody polypeptide of  claim 1  wherein the amino acid at position H51 according to the Kabat numbering convention is isoleucine.  
     
     
         18 . The antibody polypeptide of  claim 1  wherein the amino acid at position H34 according to the Kabat numbering convention is methionine.  
     
     
         19 . A composition comprising the antibody polypeptide of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         20 . A composition comprising the antibody polypeptide of  claim 1  in lyophilized form.  
     
     
         21 . A composition comprising the antibody polypeptide of  claim 1  in a form reconstituted from a lyophilized form.  
     
     
         22 . The antibody polypeptide of  claim 1  wherein members of said repertoire each comprise a binding site for a target ligand and a generic ligand.  
     
     
         23 . The antibody polypeptide of  claim 22  wherein said generic ligand is Protein A.  
     
     
         24 . The antibody polypeptide of  claim 22  wherein said polypeptide is bound to a said generic ligand.  
     
     
         25 . The antibody polypeptide of  claim 24  wherein said generic ligand is Protein A.  
     
     
         26 . An antibody polypeptide comprising a specific binding site for a human polypeptide antigen, the antibody polypeptide comprising a heavy chain variable domain that provides the binding site for said human polypeptide antigen and comprises a V H  domain sequence having a framework region encoded by human germline gene segment DP-47, wherein said variable domain is a selected member of a repertoire of antibody polypeptides, and wherein each member of said repertoire comprises a single variable domain.  
     
     
         27 . The antibody polypeptide of  claim 26  which comprises a framework region selected from the group consisting of FW1, FW2, FW3 and FW4 encoded by human germline gene segment DP-47.  
     
     
         28 . The antibody polypeptide of  claim 26  which comprises framework regions FW1, FW2, FW3 and FW4 encoded by human germline gene segment DP-47.  
     
     
         29 . An isolated antibody polypeptide having a binding site for a human polypeptide antigen, said antibody polypeptide comprising a heavy chain variable domain which provides the binding site for said human polypeptide antigen, said variable domain comprising a hypervariable loop that has the canonical structure of a hypervariable loop encoded by human germline V H  gene segment DP-47.  
     
     
         30 . The antibody polypeptide of  claim 29  wherein said hypervariable loop has the canonical structure of a hypervariable loop H1 encoded by human germline V H  gene segment DP-47.  
     
     
         31 . The antibody polypeptide of  claim 29  wherein said hypervariable loop has the canonical structure of a hypervariable loop H2 encoded by human germline V H  gene segment DP-47.  
     
     
         32 . An isolated antibody polypeptide having a binding site for a human polypeptide antigen, said antibody polypeptide comprising a heavy chain variable domain which provides the binding site for said human polypeptide antigen, said variable domain comprising a heavy chain variable domain sequence having a framework region encoded by human germline gene segment DP-47.  
     
     
         33 . The antibody polypeptide of  claim 32  which comprises a framework region selected from the group consisting of FW1, FW2, FW3 and FW4 encoded by human germline gene segment DP-47.  
     
     
         34 . The antibody polypeptide of  claim 32  which comprises framework regions FW1, FW2, FW3 and FW4 encoded by human germline gene segment DP47.  
     
     
         35 . An antibody polypeptide comprising a specific binding site for an antigen, the polypeptide comprising a light chain variable domain, wherein said variable domain provides the specific binding site for said antigen and comprises a hypervariable loop that has the canonical structure of a hypervariable loop encoded by human germline V L  gene segment DPK-9, wherein said variable domain is a selected member of a repertoire of antibody polypeptides, and wherein each member of said repertoire comprises a single variable domain.  
     
     
         36 . The antibody polypeptide of  claim 35  which comprises a hypervariable loop having the canonical structure of a hypervariable loop selected from the group consisting of hypervariable loop L1, L2 and L3 encoded by human germline V L  gene segment DPK-9.  
     
     
         37 . The antibody polypeptide of  claim 35  which comprises hypervariable loops having the canonical structures of hypervariable loops L1, L2 and L3 encoded by human germline V L  gene segment DPK-9.  
     
     
         38 . The antibody polypeptide of  claim 35  which is a Fab or F(ab′)2 polypeptide.  
     
     
         39 . The antibody polypeptide of  claim 35  which is an scFv polypeptide.  
     
     
         40 . The antibody polypeptide of  claim 35  which is a human antibody polypeptide.  
     
     
         41 . The antibody polypeptide of  claim 35  wherein said repertoire is a synthetic repertoire.  
     
     
         42 . The antibody polypeptide of  claim 35  wherein said repertoire is an immune repertoire.  
     
     
         43 . The antibody polypeptide of  claim 35  wherein members of said repertoire each comprise a binding site for a target ligand and a generic ligand.  
     
     
         44 . The antibody polypeptide of  claim 35  wherein said generic ligand is Protein L.  
     
     
         45 . The antibody polypeptide of  claim 43  wherein said polypeptide is bound to a said generic ligand.  
     
     
         46 . The antibody polypeptide of  claim 45  wherein said generic ligand is Protein L.  
     
     
         47 . The antibody polypeptide of  claim 35  wherein the members of said repertoire vary at selected positions.  
     
     
         48 . The antibody polypeptide of  claim 35  wherein the CDR1 amino acid sequence of said light chain variable domain varies from the light chain variable domain CDR1 sequence of  FIG. 2  at position L30, L31, L33 and/or L34.  
     
     
         49 . The antibody polypeptide of  claim 35  wherein the CDR1 amino acid sequence of said light chain variable domain varies from the light chain variable domain CDR1 sequence of  FIG. 2  only at position L30, L31, L33 and/or L34.  
     
     
         50 . The antibody polypeptide of  claim 35  wherein the CDR2 amino acid sequence of said light chain variable domain varies from the light chain variable domain CDR2 sequence of  FIG. 2  at position L50 and/or L53.  
     
     
         51 . The antibody polypeptide of  claim 35  wherein the CDR2 amino acid sequence of said light chain variable domain varies from the light chain variable domain CDR2 sequence of  FIG. 2  only at position L50 and/or L53.  
     
     
         52 . The antibody polypeptide of  claim 35  wherein the CDR3 amino acid sequence of said light chain variable domain varies from the light chain variable domain CDR3 sequence of  FIG. 2  at position L91, L92, L93, L94 and/or L96.  
     
     
         53 . The antibody polypeptide of  claim 35  wherein the CDR3 sequence of said light chain variable domain varies from the light chain variable domain CDR3 sequence of  FIG. 2  only at position L91, L92, L93, L94 and/or L96.  
     
     
         54 . An antibody polypeptide comprising a specific binding site for an antigen, the polypeptide comprising a light chain variable domain, wherein said variable domain provides the specific binding site for said antigen and comprises a framework region encoded by human germline V L  gene segment DPK-9, wherein said variable domain is a selected member of a repertoire of antibody polypeptides, and wherein each member of said repertoire comprises a single variable domain.  
     
     
         55 . The antibody polypeptide of  claim 54  which comprises a framework region encoded by human germline V L  gene segment DPK-9, said framework region selected from the group consisting of FW1, FW2, FW3 and FW4.  
     
     
         56 . The antibody polypeptide of  claim 54  which comprises framework regions FW1, FW2, FW3 and FW4 encoded by human germline V L  gene segment DPK-9.  
     
     
         57 . An isolated antibody polypeptide comprising a specific binding site for an antigen, said antibody polypeptide comprising a light chain variable domain which provides the binding site for said antigen, said variable domain comprising a hypervariable loop that has the canonical structure of a hypervariable loop encoded by human germline V L  gene segment DPK-9.  
     
     
         58 . An isolated antibody polypeptide comprising a specific binding site for an antigen, said polypeptide comprising a light chain variable domain polypeptide which provides the binding site for said antigen, wherein said variable domain polypeptide comprises a framework region encoded by human germline V L  gene segment DPK-9.  
     
     
         59 . The isolated antibody polypeptide of  claim 58  wherein said variable domain polypeptide comprises a framework region, selected from the group consisting of FW1, FW2, FW3 and FW4 encoded by human germline V L  gene segment DPK-9.  
     
     
         60 . The isolated antibody polypeptide of  claim 58  wherein said variable domain polypeptide comprises framework regions FW1, FW2, FW3 and FW4 encoded by human germline V L  gene segment DPK-9.