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.

Recently, the first squaramide-(SA) containing FAP inhibitor-derived radiotracers were introduced. DATA^5m.SA.FAPi and DOTA.SA.FAPi with their non-radioactive complexes showed high affinity and selectivity for FAP. After a successful preclinical study with [⁶⁸Ga]Ga-DOTA.SA.FAPi, the first patient studies were realized for both compounds. Here, we present a new squaramide-containing compound targeting FAP, based on the AAZTA⁵ chelator 1,4-bis-(carboxylmethyl)-6-[bis-(carboxymethyl)-amino-6-pentanoic-acid]-perhydro-1,4-diazepine. For this molecule (AAZTA⁵.SA.FAPi), complexation with radionuclides such as gallium-68, scandium-44, and lutetium-177 was investigated, and the in vitro properties of the complexes were characterized and compared with those of DOTA.SA.FAPi. AAZTA⁵.SA.FAPi and its derivatives labelled with non-radioactive isotopes demonstrated similar excellent inhibitory potencies compared to the previously published SA.FAPi ligands, i.e., sub-nanomolar IC₅₀ values for FAP and high selectivity indices over the serine proteases PREP and DPPs. Labeling with all three radiometals was easier and faster with AAZTA⁵.SA.FAPi compared to the corresponding DOTA analogue at ambient temperature. Especially, scandium-44 labeling with the AAZTA derivative resulted in higher specific activities. Both DOTA.SA.FAPi and AAZTA⁵.SA.FAPi showed sufficiently high stability in different media. Therefore, these FAP inhibitor agents could be promising for theranostic approaches targeting FAP.

Radiolabeled FAPI (fibroblast activation protein inhibitors) recently gained attention as widely applicable imaging and potential therapeutic compounds targeting CAF (cancer-associated fibroblasts) or DAF (disease-associated fibroblasts in benign disorders). Moreover, the use of FAPI has distinct advantages compared to FDG (e.g., increased sensitivity in regions with high glucose metabolism, no need for fasting, and rapid imaging). In this study, we wanted to evaluate the radiochemical synthesis and the clinical properties of the new CAF-targeting tracer [⁶⁸Ga]Ga-DATA^5m.SA.FAPi. The compound consists of a (radio)chemically easy to use hybrid chelate DATA.SA, which can be labeled at low temperatures, making it an interesting molecule for ‘instant kit-type’ labeling, and a squaric acid moiety that provides distinct advantages for synthesis and radiolabeling. Our work demonstrates that automatic synthesis of the FAP inhibitor [⁶⁸Ga]Ga-DATA^5m.SA.FAPi is feasible and reproducible, providing convenient access to this new hybrid chelator-based tracer. Our studies demonstrated the diagnostic usability of [⁶⁸Ga]Ga-DATA^5m.SA.FAPi for the unambiguous detection of cancer-associated fibroblasts of various carcinomas and their metastases (NSCLC, liposarcoma, parotid tumors, prostate cancer, and pancreas adenocarcinoma), while physiological uptake in brain, liver, intestine, bone, and lungs was very low.