Fibroblast activation protein inhibitors (FAPIs) labeled with gallium-68 and lutetium-177 show potential for use in the diagnosis and treatment of various cancers expressing FAP. However, 177Lu-labeled FAPIs often exhibit short tumor retention time, limiting their therapeutic applications. To improve tumor retention, we synthesized three radiolabeled dimeric FAPIs, [18F]1, [64Cu]2, and [68Ga]3. These were prepared by chelating Al[18F]F to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-l-glutamic acid (E)-(FAPI)2 and copper-64 or gallium-68 to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-E-(FAPI)2. NOTA-E-(FAPI)2 and DOTA-E-(FAPI)2 showed higher binding affinities for FAP compared with that of FAPI-04 (IC50 = 0.47 and 0.16 nM vs 0.89 nM, respectively). All radioligands were synthesized in high decay-corrected radiochemical yields (59-96%) and were stable in fetal bovine serum and phosphate-buffered saline. The more hydrophilic radioligand, [68Ga]3, was selected for cellular uptake studies, which confirmed FAP-specific uptake. Positron emission tomography imaging and ex vivo biodistribution studies in U87MG tumor-bearing mice revealed high tumor uptake of all three radioligands, with significant blocking observed after preinjection of FAPI-04. [64Cu]2 and [68Ga]3 exhibited favorable in vivo pharmacokinetics compared to those of [18F]1. Notably, [68Ga]3 showed lower normal organ uptake than did the other two radioligands, and moreover, it exhibited higher, more prolonged tumor uptake than its monomeric counterpart [68Ga]Ga-FAPI-04 over a 3 h period, suggesting its potential as a promising FAP-specific theranostic radioligand.
Keywords: PET; copper-64; dimer; fibroblast activation protein (FAP); fluorine-18; gallium-68.