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

Abstract 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 containing bifunctional DATA5m and DOTA chelators relied on UAMC1110. Results The radiopharmaceuticals DOTA.SA.FAPi and DATA5m.SA.FAPi were synthesized, labeled with gallium-68 and further characterized for in vitro stability, inhibitory efficiency, in vivo targeting properties and ex vivo biodistribution. [68Ga]Ga-DOTA.SA.FAPi and [68Ga]Ga-DATA5m.SA.FAPi showed high complexation after already 10 minutes and high stability over a period of 2 h. Affinity to FAP of DOTA.SA.FAPi and its natGa and natLu-labeled derivatives were in low nanomolar range. Comparable results were obtained for DATA5m.SA.FAPi and its natGa analogue. Additionally, all five compounds showed low affinity for the related protease PREP (high µM range). 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 and low background signal. Ex vivo biodistribution showed high tumor uptake at 60 min post injection with overall low uptake in healthy tissues. Conclusion In this work, novel PET radiotracers targeting fibroblast activation protein (FAP) 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 DATA5m 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.