Abstract 3317: Comparative biodistribution and radiotherapeutic efficacy of the fibroblast activation protein (FAP)-targeting agents FAP-2286 and FAPI-46
Authors: Zboralski Dirk , Hoehne Aileen , Bredenbeck Anne , Paschke Matthias , von Hacht Jan Lennart , Xiao Jim , Simmons Andrew D. , Osterkamp Frank , Harding Thomas , Nguyen Minh 
Published: June 2022

Abstract
Background: FAP is a membrane-bound protease under investigation as a pan-cancer target given its limited expression in normal adult tissues but high expression on cancer-associated fibroblasts. FAP-2286 and FAPI-46 are radiotracers in clinical development that target FAP through different modalities: FAP-2286 employs a peptide macrocycle, whereas FAPI-46 is a quinoline-based small molecule. Here, the biochemical and cellular properties of FAP-2286 and FAPI-46 were evaluated, as well as their in vivo biodistribution and efficacy.
Methods: FAP-2286 and FAPI-46 were assessed in biochemical and cellular assays for affinity to FAP and cellular uptake. Complexes of the compounds with gallium-68 (68Ga) or lutetium-177 (177Lu) were investigated in imaging and efficacy studies using the HEK-FAP xenograft mouse model.
Results: FAP-2286 and FAPI-46 displayed potent affinity to human FAP by surface plasmon resonance with equilibrium dissociation constants of 1.1 and 0.04 nM, respectively. In addition, FAP-2286 and FAPI-46 inhibited human FAP protease activity with IC50 of 3.2 and 1.2 nM, respectively, and competitor binding to FAP-expressing cells with IC50 of 2.7 and 1.3 nM, respectively. In a PET imaging study, 68Ga-FAP-2286 or 68Ga-FAPI-46 resulted in comparable tumor uptake at 1 hour after injection (10 MBq; 10.6 vs 10.1 percent injected dose per gram [%ID/g]). In contrast, 177Lu-FAP-2286 and 177Lu-FAPI-46 had significant differences in tumor uptake at 24 hours (30 MBq; 15.8 vs 3.8 %ID/g, respectively; P=0.001) and 72 hours (16.4 vs 1.6 %ID/g respectively; P=0.002) after dosing as observed by SPECT imaging. Consistent with the SPECT imaging data, Alexa Fluor 488-derivatized FAP-2286 was retained in cells and secluded in endosomes out to 72 hours in vitro, whereas Alexa Fluor 488-derivatized FAPI-46 levels decreased over time starting at 8 hours, despite both showing a similar level of binding and initial internalization to HEK-FAP cells. FAP-2286 and FAPI-46 labeled with 177Lu demonstrated antitumor efficacy with mean tumor volumes (MTV) of 107 and 245 mm3, respectively, on day 9 after dosing compared with 952 mm3 for vehicle control (30 MBq; P&lt;0.001). The suppressed tumor growth was maintained on day 23 for FAP-2286- but not for FAPI-46-treated animals (MTV 12 vs 1210 mm3; P&lt;0.0001). At the end of the study (day 41), all mice treated with 177Lu-FAPI-46 had reached the endpoint MTV of &gt;1500 mm³ with a median survival time (MST) of 27.5 days, whereas the MTV of mice treated with 177Lu-FAP-2286 was 106 mm3 with MST not being reached.
Conclusions: In preclinical studies, the radiotherapeutic 177Lu-FAP-2286 showed longer tumor retention, resulting in greater tumor inhibition than 177Lu-FAPI-46. The phase 1/2 LuMIERE clinical trial (NCT04939610) will evaluate FAP-2286 as a therapeutic (177Lu-FAP-2286) and imaging (68Ga-FAP-2286) agent in multiple indications.
Citation Format: Dirk Zboralski, Aileen Hoehne, Anne Bredenbeck, Matthias Paschke, Jan Lennart von Hacht, Jim Xiao, Andrew D. Simmons, Frank Osterkamp, Thomas Harding, Minh Nguyen. Comparative biodistribution and radiotherapeutic efficacy of the fibroblast activation protein (FAP)-targeting agents FAP-2286 and FAPI-46 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3317.