Detecting Functional and Accessible Folate Receptor Expression in Cancer and Polycystic Kidneys

Folate-based small molecule drug conjugates (SMDCs) are currently under development and have shown promising preclinical and clinical results against various cancers and polycystic kidney disease. Two requisites for response to a folate-based SMDC are (i) folate receptor alpha (FR alpha) protein is expressed in the diseased tissues, and (ii) FR alpha in those tissues is accessible and functionally competent to bind systemically administered SMDCs. Here we report on the development of a small molecule reporter conjugate (SMRC), called EC2220, which is composed of a folate ligand for FR alpha binding, a multilysine containing linker that can cross-link to FR alpha in the presence of formaldehyde fixation, and a small hapten (fluorescein) used for immunohistochemical detection. Data show that EC2220 produces a far greater IHC signal in FR alpha-positive tissues over that produced with EC17, a folate-fluorescein SMRC that is released from the formaldehyde-denatured FR alpha protein. Furthermore, the extent of the EC2220 IHC signal was proportional to the level of FR alpha expression. This EC2220-based assay was qualified both in vitro and in vivo using normal tissue, cancer tissue, and polycystic kidneys. Overall, EC2220 is a sensitive and effective reagent for evaluating functional and accessible receptor expression in vitro and in vivo.