Methods and reagent for preparing and using immunological agents specific for P-glycoprotein

Abstract

This invention relates to immunological reagents and methods specific for a mammalian, transmembrane protein termed Pgp, having a non-specific efflux pump activity established in the art as being a component of clinically-important multidrug resistance in cancer patients undergoing chemotherapy. The invention provides methods for developing and using immunological reagents specific for certain mutant forms of Pgp and for wild-type Pgp in a conformation associated with substrate binding or in the presence of ATP depleting agents. The invention also provides improved methods for purifying hematopoietic stems cells expressing Pgp and diagnostic and therapeutic methods for cancer cells expressing Pgp.

What is claimed is:  
     
         1 . An immunological reagent produced by introducing a cell expressing a heterologous P-glycoprotein into an animal syngeneic with the species from which the cell was derived, wherein the heterologous P-glycoprotein is in a biochemical conformation adopted in the presence of Pgp-mediated transport substrates or ATP depleting agents, and producing immune cells in the animal expressing said reagent specific for P-glycoprotein in said biochemical conformation.  
     
     
         2 . An immunological reagent according to  claim 1  that is a monoclonal antibody.  
     
     
         3 . An immunological reagent according to  claim 1 , wherein the heterologous P-glycoprotein comprises a mutant P-glycoprotein having an ATPase-specific active site in each half of the Pgp molecule comprising an amino acid residue at position 433 and 1076 of the Pgp amino acid sequence that is an amino acid residue other than lysine.  
     
     
         4 . An immunological reagent according to  claim 1 , wherein the heterologous P-glycoprotein comprises an amino acid sequence identified as Seq. I.D. No. 1.  
     
     
         5 . An immunological reagent according to  claim 1 , wherein the heterologous P-glycoprotein expressing syngeneic cell expresses a mutant P-glycoprotein wherein each of the ATPase-specific active site lysine residues of the protein are independently substituted with methionine.  
     
     
         6 . An immunological reagent according to  claim 1 , wherein the reagent is specific for P-glycoprotein in a biochemical conformation adopted in the presence of an ATP depleting agent.