Targeting fibroblast activation protein (FAP): next generation PET radiotracers using squaramide coupled bifunctional DOTA and DATA5m chelators

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.