Methods of selecting internalizing antibodies

Abstract

This invention provides methods of selecting antibodies that are internalized into target cells. The methods generally involve contacting target cells with one or more members of an antibody phage display library. The members of the phage display library are also contacted with cells of a subtractive cell line. The target cells are then washed to remove the subtractive cell line cells and members of the phage display library that are non-specifically bound or weakly bound to the target cells. The target cells are cultured under conditions where members of the phage display library can be internalized if bound to an internalizing marker and internalized members of the phage display library are then identified.

1 - 29 . Cancelled.  
     
     
         30 . A multivalent antibody phage display library, said library comprising a plurality of phage wherein said phage display, on average, at least two copies of a single-chain antibody and said library comprises a plurality of species of single-chain antibody.  
     
     
         31 . The library of  claim 30 , wherein said phage display, on average, at least 4 copies of a single chain antibody.  
     
     
         32 . The library of  claim 30 , wherein said library comprises at least 10 5  different species of single chain antibody.  
     
     
         33 . The library of  claim 30 , wherein said antibodies are encoded by a nucleic acid that is a phage vector.  
     
     
         34 . The library of  claim 30 , wherein said library is selected for members that specifically bind to an internalizing cell surface receptor.  
     
     
         35 . The library of  claim 34 , wherein said cell surface receptor is selected from the group consisting of erbB2, EGF receptor, PDGF receptor, VEGF receptor, and transferrin receptor.  
     
     
         36 . The library of  claim 30 , wherein said single-chain antibodies are single chain Fv or single-chain Fab antibodies.  
     
     
         37 . The library of  claim 30 , wherein said phage are filamentous phage.  
     
     
         38 . The library of  claim 30 , wherein said antibodies are expressed as a fusion with a PIII minor coat protein.  
     
     
         39 . The library of  claim 30 , wherein said phage express a selectable marker.  
     
     
         40 . The library of  claim 30 , wherein said selectable marker is selected from the group consisting of a fluorescent protein, an antibiotic resistance gene, and a chromagenic gene.  
     
     
         41 . The library of  claim 40 , wherein said chromagenic gene is selected from the group consisting of horse radish peroxidase, βlactamase, luciferase, and β-galactosidase.  
     
     
         42 - 47 . Cancelled.  
     
     
         48 . A kit comprising one or more containers containing a polyvalent phage display antibody library.  
     
     
         49 . The kit of  claim 48 , wherein said phage display library contains a plurality phage vectors encoding single chain antibodies in fusion with a viral coat protein.  
     
     
         50 . The kit of  claim 48 , wherein said phage display library contains a plurality of phage particles displaying, on average, at least two copies of an antibody per phage particle.