In Vitro and In Situ Activity-Based Labeling of Fibroblast Activation Protein with UAMC1110-Derived Probes

Fibroblast activation protein (FAP) is a proline-selective protease that belongs to the S9 family of serine proteases. It is typically highly expressed in the tumor microenvironment (TME) and especially in cancer-associated fibroblasts, the main cell components of the tumor stroma. The exact role of its enzymatic activity in the TME remains largely unknown. Hence, tools that enable selective, activity-based visualization of FAP within the TME can help to unravel FAP’s function. We describe the synthesis, biochemical characterization, and application of three different activity-based probes (biotin-, Cy3-, and Cy5-labeled) based on the FAP-inhibitor UAMC1110, an in-house developed molecule considered to be the most potent and selective FAP inhibitor available. We demonstrate that the three probes have subnanomolar FAP affinity and pronounced selectivity with respect to the related S9 family members. Furthermore, we report that the fluorescent Cy3- and Cy5-labeled probes are capable of selectively detecting FAP in a cellular context, making these chemical probes highly suitable for further biological studies. Moreover, proof of concept is provided for in situ FAP activity staining in patient-derived cryosections of urothelial tumors.</jats:p

Fibroblast Activation Protein (FAP) targeting homodimeric FAP inhibitor radiotheranostics : a step to improve tumor uptake and retention time

Several radiopharmaceuticals targeting fibroblast activation protein (FAP) based on the highly potent FAP inhibitor UAMC1110 are currently under investigation. Pre-clinical as well as clinical research exhibited the potential of these imaging agents. However, the monomeric small molecules seemed to have a short retention time in the tumor in combination with fast renal clearance. Therefore, our strategy was to develop homodimeric systems having two FAP inhibitors to improve residence time and tumor accumulation. The homodimers with two squaramide coupled FAP inhibitor conjugates DOTA.(SA.FAPi)(2) and DOTAGA.(SA.FAPi)(2) were synthesized and radiochemically evaluated with gallium-68. [(68)Ga]Ga-DOTAGA.(SA.FAPi)(2) was tested for its in vitro stability, lipophilicity and affinity properties. In addition, human PET/CT scans were performed for [(68)Ga]Ga-DOTAGA.(SA.FAPi)(2) with a head-to-head comparison with [(68)Ga]Ga-DOTA.SA.FAPi and [(18)F]FDG. Labeling with gallium-68 demonstrated high radiochemical yields. Inhibition measurements revealed excellent affinity and selectivity with low nanomolar IC(50) values for FAP. In PET/CT human studies, significantly higher tumor uptake as well as longer tumor retention could be observed for [(68)Ga]Ga-DOTAGA.(SA.FAPi)(2) compared to [(68)Ga]Ga-DOTA.SA.FAPi. Therefore, the introduction of the dimer led to an advance in human PET imaging indicated by increased tumor accumulation and prolonged retention times in vivo and thus, the use of dimeric structures could be the next step towards prolonged uptake of FAP inhibitors resulting in radiotherapeutic analogs of FAP inhibitors

Next generation fibroblast activation protein (FAP) targeting PET tracers : the tetrazine ligation allows an easy and convenient way to \ub9\u2078F-labeled (4-quinolinoyl)glycyl-2-cyanopyrrolidines

Abstract: Small-molecular fibroblast activation protein inhibitor (FAPI)-based tracer have been shown to be promising Positron Emission Tomography (PET) 68Ga-labeled radiopharmaceuticals to image a variety of tumors including pancreatic, breast, and colorectal cancers, among others. In this study, we developed a novel 18F-labeled FAPI derivative. [18F]6 was labeled using a synthon approach based on the tetrazine ligation. It showed subnanomolar affinity for the FAP protein and a good selectivity profile against known off-target proteases. Small animal PET studies revealed high tumor uptake and good target-to-background ratios. [18F]6 was excreted via the liver. Overall, [18F]6 showed promising characteristics to be used as a PET tracer and could serve as a lead for further development of halogen-based theranostic FAPI radiopharmaceuticals