Phage antibodies

Abstract

Peripheral blood leucocytes incubated with a semi-synthetic phage antibody library and fluorochrome-labeled CD3 and CD20 antibodies were used to isolate human single chain Fv antibodies specific for subsets of blood leucocytes by flow cytometry. Isolated phage antibodies showed exclusive binding to the subpopulation used for selection or displayed additional binding to a restricted population of other cells in the mixture. At least two phage antibodies appeared to display hithereto unknown staining patterns of B lineage cells. This approach provides a subtractive procedure to rapidly obtain human antibodies against known and novel surface antigens in their native configuration, expressed on phenotypically defined subpopulations of cells. Importantly, this approach does not depend on immunization procedures or the necessity to repeatedly construct phage antibody libraries.

1 - 8 . (Cancelled)  
     
     
         9 . A method for obtaining a phage particle comprising an antibody fragment directed against an antigen associated with the surface of target cells, the method comprising: 
 (a) providing a library of phage particles that express antibody fragments on the surface of the phage particles;    (b) incubating said library of phage particles with non-target antigens;    (c) incubating said library of phage particles with said target cells, under conditions that allow binding of the antibody fragment expressed on the surface of the phage particles to said antigen associated with said target cells;    (d) separating said target cells and phage particles bound therewith from phage particles not bound by target cells; and    (e) recovering the phage particles bound to the target cells, wherein step (c) may precede step (b).    
     
     
         10 . A method according to  claim 9 , wherein the non-target antigens are immobilized.  
     
     
         11 . A method according to  claim 10 , wherein the non-target antigens are immobilized by coating a solid surface.  
     
     
         12 . A method according to  claim 9 , wherein the non-target antigens are associated with the surface of non-target cells.  
     
     
         13 . A method according to  claim 12 , wherein the separating of said target cells and phage particles bound therewith from phage particles not bound by target cells is accomplished by flow cytometry.  
     
     
         14 . A method according to  claim 9 , further comprising isolating antibody fragments that bind to said target cells.  
     
     
         15 . A method according to  claim 13 , wherein said target cells and/or said non-target cells are detectably labeled.  
     
     
         16 . A method according to  claim 15 , wherein said detectably labeled cells are labeled with a fluorescent label.  
     
     
         17 . A method according to  claim 16 , wherein the fluorochrome-labeled antibodies are phycoerythrin (PE)-labeled, peridinin chlorophyll protein (PerCP)-labeled or fluorescein isothiocyanate (FITC)-labeled.  
     
     
         18 . A method according to  claim 9 , further comprising repeating steps (b) through (e).  
     
     
         19 . A method according to  claim 9 , wherein the library of phage particles comprises phage particles expressing Fab or single chain Fv (scFv) antibody fragments.