Comparative Study of Dimeric Fibroblast Activation Protein-Targeting Radioligands Labeled with Fluorine-18, Copper-64, and Gallium-68

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]<b>1</b>, [⁶⁴Cu]<b>2</b>, and [⁶⁸Ga]<b>3</b>. 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]<b>3</b>, 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]<b>2</b> and [⁶⁸Ga]<b>3</b> exhibited favorable in vivo pharmacokinetics compared to those of [¹⁸F]<b>1</b>. Notably, [⁶⁸Ga]<b>3</b> 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.