Identification of a PET Radiotracer for Imaging of the Folate Receptor-α: A Potential Tool to Select Patients for Targeted Tumor Therapy

The aim of this study was to identify a folate receptor-α (FRα)–selective PET agent potentially suitable for the selection of patients who might profit from FRα-targeted therapies. The 6R and 6S isomers of (18)F-aza-5-methyltetrahydrofolate (MTHF) were assessed regarding their binding to FRα and FRβ, expressed on cancer and inflammatory cells, respectively, and compared with (18)F-AzaFol, the folic acid–based analog. Methods: FR selectivity was investigated using FRα-transfected (RT16) and FRβ-transfected (D4) CHO cells. The cell uptake of (18)F-folate tracers was investigated, and receptor-binding affinities were determined with the nonradioactive analogs. In vitro autoradiography of the (18)F-folate tracers was performed using RT16 and D4 tissue sections. Biodistribution studies and PET/CT imaging of the radiotracers were performed on mice bearing RT16 and D4 xenografts. Results: The uptake of (18)F-6R-aza-5-MTHF was high when using RT16 cells (62% ± 10% of added activity) but much lower when using D4 cells (5% ± 2%). The FRα selectivity of (18)F-6R-aza-5-MTHF was further demonstrated by its approximately 43-fold higher binding affinity to FRα (half-maximal inhibitory concentration [IC(50)], 1.8 ± 0.1 nM) than to FRβ (IC(50), 77 ± 27 nM). The uptake of (18)F-6S-aza-5-MTHF and (18)F-AzaFol was equal in both cell lines (52%–70%), with similar affinities to FRα (IC(50), 2.1 ± 0.4 nM and 0.6 ± 0.3 nM, respectively) and FRβ (0.8 ± 0.2 nM and 0.3 ± 0.1 nM, respectively). The autoradiography signal obtained with (18)F-6R-aza-5-MTHF was 11-fold more intense for RT16 than for D4 tissue sections. Biodistribution data showed high uptake of (18)F-6R-aza-5-MTHF in RT16 xenografts (81% ± 20% injected activity per gram [IA]/g 1 h after injection) but significantly lower accumulation in D4 xenografts (7.3% ± 2.1% IA/g 1 h after injection), which was also visualized using PET. The uptake of (18)F-6S-aza-5-MTHF and (18)F-AzaFol was similar in RT16 (53% ± 10% IA/g and 45% ± 2% IA/g, respectively) and D4 xenografts (77% ± 10% IA/g and 52% ± 7% IA/g, respectively). Conclusion: This study demonstrated FRα selectivity for (18)F-6R-aza-5-MTHF but not for (18)F-6S-aza-5-MTHF or (18)F-AzaFol. This characteristic, together with its favorable tissue distribution, makes (18)F-6R-aza-5-MTHF attractive for clinical translation to enable detection of FRα-positive cancer while preventing undesired accumulation in FRβ-expressing inflammatory cells.