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

Abstract

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, Ga-AV01017, Ga-AV01030, and Ga-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.

Novel 68Ga-Labeled Pyridine-Based Fibroblast Activation Protein-Targeted Tracers with High Tumor-to-Background Contrast

“… Inhibition of FAP enzymatic activity on Suc-Gly-Pro-AMC substrate by Ga-AV02053 and Ga-AV02070, and comparison with the data obtained previously using Ga-FAPI-04 [ 31 ]. The enzymatic activity represents the fluorescence of AMC which was cleaved by human FAP enzyme.…”

“…The calculated IC 50 values for Ga-AV02053 and Ga-AV02070 were 187 ± 52.0 and 17.1 ± 4.60 nM, respectively. For comparison, the previously reported IC 50 value for Ga-FAPI-04 under the same assay conditions was 1.03 ± 0.44 nM ( Figure 2 ) [ 31 ].…”

“…Consistent with the predictions from their binding affinity and smaller molecular size of pharmacophores, PET imaging and biodistribution data ( Figure 3 , Figure 4 and Figure 5 and Table S4 ) revealed that the pyridine-based tracers, [ 68 Ga]Ga-AV02070 (7.93 ± 1.88%ID/g) and [ 68 Ga]Ga-AV02053 (5.60 ± 1.12%ID/g), have a lower tumor uptake than the previously reported value of the quinoline-based [ 68 Ga]Ga-FAPI-04 (12.5 ± 2.00%ID/g) using the same HEK293T:hFAP tumor model [ 31 ]. However, the pyridine-based tracers have a lower uptake in muscle and bone, which are the two common off-target organs of FAP-targeted tracers, as observed in the quinoline-based [ 68 Ga]Ga-FAPI-04 ( Table S4 ).…”

“…The HEK293T:hFAP cells generated in our lab [ 31 ] were cultured in DMEM GlutaMAX™ medium supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37 °C in a Panasonic Healthcare (Tokyo, Japan) MCO-19AIC humidified incubator containing 5% CO 2 . Cells were grown until 80–90% confluence and washed with sterile phosphate-buffered saline (PBS, pH 7.4) and collected after 1 min trypsinization.…”

“…Therefore, we designed [ 68 Ga]Ga-AV02070 to compare with [ 68 Ga]Ga-AV02053. The potential of [ 68 Ga]Ga-AV02053 and [ 68 Ga]Ga-AV02070 for cancer imaging was evaluated by in vitro enzymatic assay, PET imaging, and ex vivo biodistribution studies using a preclinical tumor model, HEK293T:hFAP [ 31 ]. The results were then compared with those previously obtained using [ 68 Ga]Ga-FAPI-04 [ 31 ].…”