Abstract
Triple-negative breast cancer (TNBC) is characterized by considerable heterogeneity and a poor response to chemotherapy. While folate receptor alpha (FRα) has attracted growing interest as a therapeutic target due to its frequent overexpression in numerous epithelial-derived tumors, we sought to visually evaluate its specific expression profile in TNBC. Therefore, we first evaluated FRα expression in 176 clinical TNBC specimens. The vast majority (84%) of samples showed no FRα expression, with the remainder showing low to moderate positivity (1+ to 3+), and no significant correlation was found with standard clinicopathological features. Given the low and variable FRα expression in TNBC, we developed an immunoPET probe, (89)Zr-DFO-Mirvetuximab, for noninvasive quantification. This probe was synthesized by conjugating the anti-FRα antibody Mirvetuximab with the chelator desferrioxamine (DFO), followed by (89)Zr-labeling, exhibiting high radiochemical purity (>95%) and exceptional stability. Cell uptake assays revealed time-dependent accumulation, which was blocked by excess Mirvetuximab, demonstrating FRα-specific targeting. Binding affinity measurement confirmed high affinity for FRα, with a dissociation constant (K (d)) of 27.46 nM. We further validated the probe in vivo using micro-PET/CT imaging and biodistribution studies in mice bearing CAL51 (FRα-high) and MDA-MB-231 (FRα-low) tumors. The results demonstrated significantly higher uptake in CAL51 models compared to MDA-MB-231 models (17.17 ± 2.24%ID/g vs 3.79 ± 1.15%ID/g, P < 0.001), which was further confirmed by subsequent immunohistochemical analysis. In conclusion, (89)Zr-DFO-Mirvetuximab holds significant potential for noninvasively identifying TNBC patients with sufficient FRα expression for targeted therapies like Mirvetuximab soravtansine, thereby supporting improved patient stratification and treatment response monitoring.