Methods and Reagents 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 identifying and characterizing anticancer compounds.

1 . A method for characterizing binding of a test compound to P-glycoprotein expression in a mammalian cell, the method comprising the steps of:
 (a) incubating a first mammalian cell expressing P-glycoprotein in the presence of a first P-glycoprotein binding compound having a first binding affinity for P-glycoprotein;   (b) incubating a second mammalian cell expressing P-glycoprotein in the presence of the test compound;   (c) reacting the first mammalian cell and the second mammalian cell with an immunological reagent specific for P-glycoprotein in a biochemical conformation adopted in the presence of a P-glycoprotein substrate; and   (d) comparing binding of the immunological reagent to the first mammalian cell with binding of the immunological reagent to the second mammalian cell.   
     
     
         2 . The method of  claim 1  wherein the immunological reagent is a monoclonal antibody specific for P-glycoprotein in a biochemical conformation adopted in the presence of an ATP-depleting agent. 
     
     
         3 . The monoclonal antibody of  claim 2  that is UIC2 (A.T.C.C. Accession No. HB11027). 
     
     
         4 . The method of  claim 1 , wherein binding of the immunological reagent is increased in the presence of the P-glycoprotein substrate. 
     
     
         5 . The method of  claim 1  wherein the immunological reagent is detectably-labeled. 
     
     
         6 . The method of  claim 5  wherein the detectable label is a fluorescent label. 
     
     
         7 . The method of  claim 1  wherein binding of the immunological reagent is increased in the presence of the P-glycoprotein substrate and wherein enhanced binding of the fluorescently-labeled immunological reagent is detected by fluorescence-activated cell sorting.