One Ligand, Two Imaging Modalities: [99mTc]Tc-FAPI-74 and [18F]AlF-FAPI-74

Fibroblast activation protein (FAP) is highly expressed on cancer-associated fibroblasts, making it an attractive target for oncological imaging and therapy. FAPI-74, a FAP inhibitor (FAPi) in phase II clinical evaluation with 18F as a PET tracer, has exhibited high tumour uptake and specificity. Development of SPECT analogue [99mTc]Tc-FAPI-74 offers advantages of lower cost and accessibility through established SPECT infrastructure. Herein, we report the radiolabeling and characterization of FAPI-74 with 99mTc and [18F]AlF, and comparative evaluation of their binding properties and biodistribution in FAP-positive and FAP-negative xenograft mouse models. FAPI-74 was radiolabeled with [99mTc]Tc-tricarbonyl, achieving radiochemical conversion (RCC) of 20% and 0.32 GBq/µmol molar activity (Am). Comparatively, [18F]AlF labeling resulted in 75% RCC, and 3.33 GBq/µmol Am. Both tracers exhibited >99% radiochemical purity. In vitro binding to FAP+ cells was comparable between [99mTc]Tc-FAPI-74 and [18F]AlF-FAPI-74 (29.9 ± 4.7% vs. 31.4 ± 11.0% binding and KD values of 14.6 nM vs. 3.1 nM). In vivo, tumour uptake in HT29 xenografts was similar (2.14 ± 0.29 vs. 1.59 ± 0.12 %ID/g) with a %ID/g of <0.5 in the blocked group of both ligands. However, in HEK-293-FAP+ xenografts, tumour uptake was markedly lower for [99mTc]Tc-FAPI-74 (3.17 ± 0.26 vs. 25.73 ± 4.49 %ID/g). [99mTc]Tc-FAPI-74 demonstrates comparable in vitro performance to [18F]AlF-FAPI-74, with similar binding and KD values, but its higher lipophilicity limits its clinical competitiveness due to low tumour uptake of [99mTc]Tc-FAPI-74. Nonetheless, its FAP-specific uptake in mouse models, together with the availability of FAPI-74 and facile 99mTc radiolabeling, highlights its potential for imaging tumours outside the hepatobiliary system. Future SPECT FAP tracers should improve hydrophilicity to minimize hepatobiliary clearance and improve tumour specificity.