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 . A method of selecting antibody binding moieties that are internalized into target cells, said method comprising:
 i) contacting one or more of said target cells with one or more members of a polyvalent antibody phage display library;   ii) culturing said target cells under internalizing conditions; and   iii) identifying members of said polyvalent antibody phage display library internalized into the one or more of said target cells.   
     
     
         2 . The method of  claim 1 , wherein said polyvalent phage display library comprises:
 a) a phage display library in which each member phase displays, on average, at least one copy of an antibody-phage coat protein fusion product comprising two or more binding domains, each said domain including a variable heavy chain region and a variable light chain region of an antibody; or   b) a phase display library in which each member phage displays, at average, two or more copies of an antibody-phage coat protein fusion product comprising a variable heavy chain region and a variable light chain region of an antibody.   
     
     
         3 . The method of  claim 2 , wherein said antibody phage display library displays single chain antibody Fv regions. 
     
     
         4 . The method of  claim 1 , wherein said identifying comprises recovering internalized phage and optionally repeating steps (i) through (iii) to further select for internalizing binding moieties. 
     
     
         5 . The method of  claim 4 , wherein said recovering comprises:
 (a) lysing said target cells to release internalized phage; and   (b) infecting a bacterial host with said internalized phage to produce phage for a subsequent round of selection.   
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 1 , further comprising the step of contacting members of said polyvalent antibody phase display library with cells of a subtractive cell line. 
     
     
         8 . The method of  claim 7 , wherein said cells of a subtractive cell line are present in at least 2-fold excess over said target cells. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein step (ii) comprises culturing the target cells at a temperature of about 35° C. to about 37° C. and a pH from about 6 to about 8. 
     
     
         12 . (canceled) 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1 , wherein said target cells are selected from the group consisting of solid tumor cells, members of a cDNA expression library, cells that overexpress a cytokine receptor, cells that overexpress a growth factor receptor, metastatic cells, cells of a transformed cell line, cells transformed with a gene or cDNA encoding a specific surface target receptor, and neoplastic cells derived from outside a solid tumor. 
     
     
         16 . The method of  claim 7 , wherein said cells of a subtractive cell line are selected from the same tissue type as the target cells. 
     
     
         17 . The method of  claim 7 , wherein said cells of a subtractive cell line are selected from the group consisting of fibroblasts, monocytes, stem cells, and lymphocytes. 
     
     
         18 - 50 . (canceled) 
     
     
         51 . The method of  claim 4 , wherein said recovering comprises removing polyvalent antibody phage display library members that are not internalized by the one or more target cells. 
     
     
         52 . The method of  claim 51 , wherein said removing comprises washing the target cells with a strong wash. 
     
     
         53 . The method of  claim 51 , wherein said removing comprises removing the polyvalent antibody phage display library members bound to an extracellular matrix of the target cells. 
     
     
         54 . The method of  claim 51 , wherein said removing comprises trypsinization of the target cells. 
     
     
         55 . The method of  claim 7  wherein said cells of a subtractive cell line are present in at least 2-fold excess over said target cells. 
     
     
         56 . The method of  claim 7 , wherein said cells of a subtractive cell line comprise live cells. 
     
     
         57 . The method of  claim 7 , wherein the one or more target cells comprise the cells of a subtractive cell line induced to express on their surface a target epitope, whereby the internalizing antibody moieties that bind to said target epitope are selected. 
     
     
         58 . The method of  claim 57 , wherein the target cells comprise cells of a subtractive cell line transformed with a nucleic acid that encodes said target epitope.