Characterization of the Human Folate Receptor Alpha Via Novel Antibody-Based Probes

Abstract

Folate receptor alpha (FRA) is a cell surface protein whose aberrant expression in malignant cells has resulted in its pursuit as a therapeutic target and marker for diagnosis of cancer. The development of immune-based reagents that can reproducibly detect FRA from patient tissue processed by varying methods has been difficult due to the complex post-translational structure of the protein whereby most reagents developed to date are highly structure-sensitive and have resulted in equivocal expression results across independent studies. The aim of the present study was to generate novel monoclonal antibodies (mAbs) using modified full length FRA protein as immunogen in order to develop a panel of mAbs to various, non-overlapping epitopes that may serve as diagnostic reagents able to robustly detect FRA-positive disease. Here we report the development of a panel of FRA-specific mAbs that are able to specifically detect FRA using an array of diagnostic platforms and methods. In addition, the methods used to develop these mAbs and their diverse binding properties provide additional information on the three dimensional structure of FRA in its native cell surface configuration.

Folate Receptor Alpha Expression in Lung Cancer: Diagnostic and Prognostic Significance

“…The mechanisms of increased FRA expression in cancer and its potential effect on prognosis are not well understood. While folic acid/vitamin B9 is essential to all mammalian cells, given its critical role in 1-carbon metabolism and methylation of nucleotides, lipids and proteins, FRA is apparently not required since its expression in normal tissues is highly restricted and its function more distinct than the ubiquitously expressed reduced folate receptor (RFC) (19). FRA has been clearly demonstrated to be essential during embryonic development but its role in some terminally differentiated adult tissues is, at best, speculative.…”

“…The mean (± SEM) M-scores for adenocarcinoma and squamous cell carcinoma samples stained with MAb 26B3 were 19 (± 5.54), respectively (p<0.0001). The M-score for adenocarcinoma samples was also significantly higher when compared against all other lung cancer histologic types.…”

“…After washing in TBST as above, Background Sniper (Biocare Medical) serum-free universal blocking reagent was applied for 10min at RT. Slides were then incubated with purified MAb 26B3.F2 [19] hematoxylin (Dako) for 2min, all incubations being performed at RT. Scoring for staining was performed by a single board-certified pathologist, using customary scoring for intensity and the percent of the tumor stained at each intensity.…”

“…Detection of FRA on formalinfixed paraffin embedded (FFPE) tissue sections via immunohistochemistry (IHC) requires highly sensitive and specific reagents. The development and characterization of novel, high-affinity FRA-specific antibodies that can be used in multiple diagnostic platforms, including IHC, has recently been described [19]. One unique clone, monoclonal antibody 26B3.F2 (MAb 26B3) displayed high-affinity FRA binding and the ability to recognize FRA in both its native and denatured forms [19].…”

The antitumor activity of the human FOLR1-specific monoclonal antibody, farletuzumab, in an ovarian cancer mouse model is mediated by antibody-dependent cellular cytotoxicity

“…34 Surface plasmon resonance analysis was conducted on a Biacore 3000 instrument (GE Healthcare Biosciences) and experiments were repeated twice. WT-FRL and MUT-FRL were diluted to 3 µg/mL using HEPES-buffer saline containing EDTA and P-10 non-ionic detergent.…”