Synthesis and Preclinical Evaluation of Three Novel ⁶⁸Ga-Labeled Bispecific PSMA/FAP-Targeting Tracers for Prostate Cancer Imaging

Tumor heterogeneity limits the efficacy and reliability of monospecific radiopharmaceuticals in prostate cancer diagnosis and therapy. To overcome this limitation and improve lesion detection sensitivity, we developed and evaluated three bispecific radiotracers that can target both prostate-specific membrane antigen (PSMA) and fibroblast activation protein (FAP), which are the two key proteins overexpressed in prostate cancer. Three FAP-targeting ligands with various linker lengths were synthesized through multistep organic synthesis, and then connected to the PSMA-targeting motif. IC₅₀(PSMA) and IC₅₀(FAP) values of Ga-complexed bispecific ligands, ⁶⁸Ga-AV01017, ⁶⁸Ga-AV01030, and ⁶⁸Ga-AV01038 were 25.2–71.6 and 1.25–2.74 nM, respectively. The uptake values in PSMA-expressing LNCaP tumor xenografts were 4.38 ± 0.55, 5.17 ± 0.51, and 4.25 ± 0.86 %ID/g for [⁶⁸Ga]Ga-AV01017, [⁶⁸Ga]Ga-AV01030, and [⁶⁸Ga]Ga-AV01038, respectively, which were lower than the monospecific PSMA-targeting tracer [⁶⁸Ga]Ga-HTK03041 (23.1 ± 6.11 %ID/g). The uptake values in FAP-expressing HEK293T:hFAP tumor xenografts were 2.99 ± 0.37, 3.69 ± 0.81, 3.64 ± 0.83 %ID/g for [⁶⁸Ga]Ga-AV01017, [⁶⁸Ga]Ga-AV01030, and [⁶⁸Ga]Ga-AV01038, respectively, which were also lower than the monospecific FAP-targeting tracer, [⁶⁸Ga]Ga-FAPI-04 (12.5 ± 2.00 %ID/g). We observed that the bispecific tracers had prolonged blood retention, in which tracers with a longer linker tend to have a higher blood uptake and lower tumor uptake. Further investigations are needed to optimize the linker selection to generate promising bispecific PSMA/FAP-targeting tracers for prostate cancer imaging.