Methods of high-throughput screening for internalizing antibodies

Abstract

This invention provides methods of identifying ligands that are internalized into a cell. The methods typically involve i) contacting the cell with a reporter non-covalently coupled to a ligand; ii) dissociating the reporter from the ligand and removing dissociated reporter from the surface of the cell; and iii) detecting the reporter within said cell (if any is present) where the presence of the reporter within said cell indicates that the ligand binds to an internalizing receptor and is internalized.

1 - 43 . (canceled)  
     
     
         44 . A construct for screening a cell for an internalizing receptor, said construct comprising a ligand non-covalently coupled to an effector through an epitope tag.  
     
     
         45 . The construct of  claim 44 , wherein said ligand is a peptide.  
     
     
         46 . The construct of  claim 44 , wherein said ligand is selected from the group consisting of an scFv, an Fv, an Fab, a monoclonal antibody, a cytokine, an enzyme, a hormone, and a growth factor.  
     
     
         47 . The construct of  claim 44 , wherein said ligand is a member of a combinatorial library.  
     
     
         48 . The method of  claim 74 , wherein said phage display library uses a filamentous phage.  
     
     
         49 . The construct of  claim 44 , wherein said epitope tag is selected from the group consisting of a His-tag, a Flag-tag, an HA-tag, a myc-tag, and a DYKDDDDK (SEQ ID NO:1) epitope.  
     
     
         50 . The construct of  claim 44 , wherein the effector comprises a nanoparticle or a liposome.  
     
     
         51 . The construct of  claim 44 , wherein said epitope tag is a hexahistidine (His 6 ) tag and said effector is a liposome comprising a nitrilotriacetic acid (NTA) lipid or an iminodioacetic acid (IDA) lipid.  
     
     
         52 . The construct of  claim 44 , wherein said ligand is an antobody and said epitope tag is attached to said antibody through a covalent linkage to protein A or protein G.  
     
     
         53 . The construct of  claim 44 , wherein said construct is polyvalent for said ligand.  
     
     
         54 - 61 . (canceled)  
     
     
         62 . A metal chelating lipid composition comprising a lipid, a hydrophilic polymer, and a metal chelation group attached to said hydrophilic polymer.  
     
     
         63 . The metal chelating lipid composition of  claim 62 , wherein said metal chelation group is NTA or IDA.  
     
     
         64 . The metal chelating lipid of  claim 62 , wherein said hydrophilic polymer comprises polynucleotide(ethylene glycol).  
     
     
         65 . The metal chelating lipid of  claim 62 , wherein said lipid comprises distearoylphosphatidylethanolamine (DSPE).  
     
     
         66 - 68 . (canceled)  
     
     
         69 . A composition for delivering an agent to a cell, the composition comprising 
 the construct of  claim 44 , wherein the effector comprises the metal chelation lipid composition of  claim 62 , wherein the metal chelation group of the metal chelation lipid composition is capable of forming a chelation bond with the epitope tag of the construct;    a ligand comprising said epitope tag where said ligand binds and is optionally internalized by a cell; and    an agent associated with said metal chelation lipid composition.    
     
     
         70 . The composition of  claim 69 , wherein said metal chelation lipid composition comprises a liposome and said liposome contains or is complexed with said agent.  
     
     
         71 . The composition of  claim 69 , wherein said hydrophilic polymer of the metal chelation lipid composition comprised poly (ethylene glycol).  
     
     
         72 . The composition of  claim 69 , wherein said lipid of the metal chelation lipid composition comprises distearoylphosphatidylethanolamine (DSPE).  
     
     
         73 . The composition of  claim 69 , wherein the agent comprises a reporter, a cytotoxin, a drug, or a nucleic acid.  
     
     
         74 . The construct of  claim 47 , wherein said combinatorial library comprises a combinatorial chemical library, a recombinant library, or a phage display library.  
     
     
         75 . The construct of  claim 44 , wherein the effector comprises a reporter selected from the group consisting of an enzyme, a colorimetric label, a fluorescent label, a luminescent label, and a radioactive label.