Comparative\nStudy of Dimeric Fibroblast Activation\nProtein-Targeting Radioligands Labeled with Fluorine-18, Copper-64,\nand Gallium-68

Fibroblast activation protein inhibitors (FAPIs) labeled\nwith gallium-68\nand lutetium-177 show potential for use in the diagnosis and treatment\nof various cancers expressing FAP. However, <sup>177</sup>Lu-labeled\nFAPIs often exhibit short tumor retention time, limiting their therapeutic\napplications. To improve tumor retention, we synthesized three radiolabeled\ndimeric FAPIs, [<sup>18</sup>F]<b>1</b>, [<sup>64</sup>Cu]<b>2</b>, and [<sup>68</sup>Ga]<b>3</b>. These were prepared\nby chelating Al[<sup>18</sup>F]F to 1,4,7-triazacyclononane-1,4,7-triacetic\nacid (NOTA)-l-glutamic acid (E)-(FAPI)<sub>2</sub> and copper-64\nor gallium-68 to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic\nacid (DOTA)-E-(FAPI)<sub>2</sub>. NOTA-E-(FAPI)<sub>2</sub> and DOTA-E-(FAPI)<sub>2</sub> showed higher binding affinities for FAP compared with that\nof FAPI-04 (IC<sub>50</sub> = 0.47 and 0.16 nM vs 0.89 nM, respectively).\nAll radioligands were synthesized in high decay-corrected radiochemical\nyields (59–96%) and were stable in fetal bovine serum and phosphate-buffered\nsaline. The more hydrophilic radioligand, [<sup>68</sup>Ga]<b>3</b>, was selected for cellular uptake studies, which confirmed FAP-specific\nuptake. Positron emission tomography imaging and ex vivo biodistribution\nstudies in U87MG tumor-bearing mice revealed high tumor uptake of\nall three radioligands, with significant blocking observed after preinjection\nof FAPI-04. [<sup>64</sup>Cu]<b>2</b> and [<sup>68</sup>Ga]<b>3</b> exhibited favorable in vivo pharmacokinetics compared to\nthose of [<sup>18</sup>F]<b>1</b>. Notably, [<sup>68</sup>Ga]<b>3</b> showed lower normal organ uptake than did the other two\nradioligands, and moreover, it exhibited higher, more prolonged tumor\nuptake than its monomeric counterpart [<sup>68</sup>Ga]Ga-FAPI-04\nover a 3 h period, suggesting its potential as a promising FAP-specific\ntheranostic radioligand.