Background: Fibroblast activation protein (FAP) is a proline selective serine protease that is overexpressed in tumor stroma and in lesions of many other diseases that are characterized by tissue remodeling. In 2014, a most potent FAP-inhibitor (referred to as UAMC1110) with low nanomolar FAP-affinity and high selectivity toward related enzymes such as prolyl oligopeptidase (PREP) and the dipeptidyl-peptidases (DPPs): DPP4, DPP8/9 and DPP2 were developed. This inhibitor has been adopted recently by other groups to create radiopharmaceuticals by coupling bifunctional chelator-linker systems. Here, we report squaric acid (SA) containing bifunctional DATA^5m and DOTA chelators based on UAMC1110 as pharmacophor. The novel radiopharmaceuticals DOTA.SA.FAPi and DATA^5m.SA.FAPi with their non-radioactive derivatives were characterized for in vitro inhibitory efficiency to FAP and PREP, respectively and radiochemical investigated with gallium-68. Further, first proof-of-concept in vivo animal study followed by ex vivo biodistribution were determined with [⁶⁸Ga]Ga-DOTA.SA.FAPi. Results: [⁶⁸Ga]Ga-DOTA.SA.FAPi and [⁶⁸Ga]Ga-DATA^5m.SA.FAPi showed high complexation > 97% radiochemical yields after already 10 min and high stability over a period of 2 h. Affinity to FAP of DOTA.SA.FAPi and DATA^5m.SA.FAPi and its ^natGa and ^natLu-labeled derivatives were excellent resulting in low nanomolar IC₅₀ values of 0.7–1.4 nM. Additionally, all five compounds showed low affinity for the related protease PREP (high IC₅₀ with 1.7–8.7 μM). First proof-of-principle in vivo PET-imaging animal studies of the [⁶⁸Ga]Ga-DOTA.SA.FAPi precursor in a HT-29 human colorectal cancer xenograft mouse model indicated promising results with high accumulation in tumor (SUV_mean of 0.75) and low background signal. Ex vivo biodistribution showed highest uptake in tumor (5.2%ID/g) at 60 min post injection with overall low uptake in healthy tissues. Conclusion: In this work, novel PET radiotracers targeting fibroblast activation protein were synthesized and biochemically investigated. Critical substructures of the novel compounds are a squaramide linker unit derived from the basic motif of squaric acid, DOTA and DATA^5m bifunctional chelators and a FAP-targeting moiety. In conclusion, these new FAP-ligands appear promising, both for further research and development as well as for first human application.

Hardly any new tracers attracted more attention in nuclear medicine in the last couple of years than radiolabeled fibroblast activation protein inhibitors (FAPi’s). Molecules targeting cancer-associated fibroblasts (CAFs) or disease-associated fibroblasts in benign disorders (DAFs) gave rise to a new class of radiopharmaceuticals widely applicable for imaging and with the desired use as therapeutic compounds. Despite displaying benefits in diagnostic sensitivity over FDG, most FAP-targeting compounds in today’s clinical routine continue to lack therapeutic utility due to short tumor retention. In this study, we evaluated 3BP-3940, specifically designed for achieving prolonged tumor retention and remarkably low uptake in healthy tissues. We herein present the automated manufacturing of gallium-68 (Ga-68) and lutetium-177 (Lu-177)-labeled 3BP-3940, their respective in vitro stability, validation of an automated production process, and validation of an analytical HPLC method for quality control. Finally, we give a first insight into the clinical utility of the two compounds.