Compositions and methods for the identification and isolation of cell-membrane protein specific binding moieties

Abstract

Disclosed are methods of identifying binding moieties that recognize antigens displayed on cells, such as membrane proteins or recombinant proteins that display eptiopes on the surface of cells. Binding moieties capable of binding membrane proteins can be difficult to obtain because these proteins can depend on their native environments for structural integrity. In some methods scFv phage display libraries are panned against whole cells expressing a membrane protein in an emulsion. Certain methods further permit discrimination of binding moieties according to their affinity or avidity for a target. This approach allows rapid identification of cell surface epitope specific antibodies for research, diagnostics, and immunotherapeutics.

What is claimed is: 
     
         1 - 14 . (canceled) 
     
     
         15 . An emulsion comprising a plurality of droplets, wherein said droplets comprise:
 (i) a plurality of antigen carriers displaying a cell-membrane bound epitope, wherein said antigen carriers comprise animal cells; and   (ii) a plurality of binding moiety carriers, wherein the plurality of said binding moiety carriers comprise viruses encoding and displaying the binding moieties, the viruses being released into said droplets by virus-transduced bacteria.   
     
     
         16 . The emulsion of  claim 15 , wherein, within said droplets, one or more of said antigen carriers displaying said cell-membrane bound epitope contacts a plurality of said binding moiety carriers; and
 wherein, if an individual binding moiety is specific to said antigen carrier present in the same droplet, said contacting results in a complex comprising at least one binding moiety bound to a membrane of an intact binding moiety carrier and at least one antigen bound to a membrane of an intact antigen carrier.   
     
     
         17 . The emulsion of  claim 15 , wherein said binding moiety carrier is an M13 bacteriophage, a lambda phage, a T4 phage, or a T7 phage. 
     
     
         18 . The emulsion of  claim 15 , wherein said binding moiety comprises a DARPin, FN3 (fibronectin repeats), Fork-head associate protein, scFv, Fab, or other antibody-derived protein. 
     
     
         19 . The emulsion of  claim 15 , wherein said virus-transduced bacteria are phage-transduced bacteria. 
     
     
         20 . The emulsion of  claim 19 , wherein said phage-transduced bacteria are phage-transduced  E. coli.    
     
     
         21 . The emulsion of  claim 15 , wherein said cell-membrane bound epitope is recombinantly expressed by said animal cells or is natively expressed by said animal cells. 
     
     
         22 . The emulsion of  claim 15 , wherein said plurality of binding moiety carriers was generated by contacting a plurality of animal cells not having said cell-membrane bound epitope with a plurality of binding moieties and washing said animal cells not having said cell-membrane bound epitope, thereby forming a mixture of binding moiety carriers that failed to bind said animal cells not having said cell-membrane bound epitope, and isolating said mixture of binding moiety carriers, thereby generating said plurality of binding moieties. 
     
     
         23 . The emulsion of  claim 15 , wherein the emulsion is a monodispersed emulsion. 
     
     
         24 . The emulsion of  claim 15 , wherein the emulsion is a heterodispersed emulsion. 
     
     
         25 . The emulsion of  claim 15 , wherein the emulsion is an oil and water emulsion, or wherein the emulsion is a perfluorcarbon oil emulsion. 
     
     
         26 . The emulsion of  claim 15 , wherein each droplet comprises, or comprises on average, 0-10 antigen carriers. 
     
     
         27 . The emulsion of  claim 26 , wherein a given droplet comprises 0, 1, 2, or 3 antigen carriers. 
     
     
         28 . The emulsion of  claim 15 , wherein each droplet comprises, or comprises on average, 0-1000 binding moiety carriers. 
     
     
         29 . The emulsion of  claim 15 , wherein said emulsion comprises a clonal population of binding moiety carriers. 
     
     
         30 . The emulsion of  claim 29 , wherein a given droplet includes 0, 1, 2, or 3 bacterial cells that each secrete a clonal population of binding moiety carriers. 
     
     
         31 . The emulsion of  claim 15 , wherein said animal cell is a mammalian cell. 
     
     
         32 . The emulsion of  claim 31 , wherein said mammalian cell is a human cell.