Development of FAPI Tetramers to Improve Tumor Uptake and Efficacy of FAPI Radioligand Therapy

Yizhen Pang, Liang Zhao, Jianyang Fang, Jianhao Chen, Lingxin Meng, Long Sun, Hua Wu, Zhide Guo, Qin Lin, Haojun Chen, Yizhen Pang, Liang Zhao, Jianyang Fang, Jianhao Chen, Lingxin Meng, Long Sun, Hua Wu, Zhide Guo, Qin Lin, Haojun Chen

Abstract

Radiolabeled fibroblast activation protein (FAP) inhibitors (FAPIs) have shown promise as cancer diagnostic agents; however, the relatively short tumor retention of FAPIs may limit their application in radioligand therapy. In this paper, we report the design, synthesis, and evaluation of a FAPI tetramer. The aim of the study was to evaluate the tumor-targeting characteristics of radiolabeled FAPI multimers in vitro and in vivo, thereby providing information for the design of FAP-targeted radiopharmaceuticals based on the polyvalency principle. Methods: FAPI tetramers were synthesized on the basis of FAPI-46 and radiolabeled with 68Ga, 64Cu, and 177Lu. In vitro FAP-binding characteristics were identified using a competitive cell-binding experiment. To evaluate their pharmacokinetics, small-animal PET, SPECT, and ex vivo biodistribution analyses were performed on HT-1080-FAP and U87MG tumor-bearing mice. In addition, the 2 tumor xenografts received radioligand therapy with 177Lu-DOTA-4P(FAPI)4, and the antitumor efficacy of the 177Lu-FAPI tetramer was evaluated and compared with that of the 177Lu-FAPI dimer and monomer. Results: 68Ga-DOTA-4P(FAPI)4 and 177Lu-DOTA-4P(FAPI)4 were highly stable in phosphate-buffered saline and fetal bovine serum. The FAPI tetramer exhibited high FAP-binding affinity and specificity both in vitro and in vivo. 68Ga-, 64Cu-, and 177Lu-labeled FAPI tetramers exhibited higher tumor uptake, longer tumor retention, and slower clearance than FAPI dimers and FAPI-46 in HT-1080-FAP tumors. The uptake (percentage injected dose per gram) of 177Lu-DOTA-4P(FAPI)4, 177Lu-DOTA-2P(FAPI)2, and 177Lu-FAPI-46 in HT-1080-FAP tumors at 24 h was 21.4 ± 1.7, 17.1 ± 3.9, and 3.4 ± 0.7, respectively. Moreover, 68Ga-DOTA-4P(FAPI)4 uptake in U87MG tumors was approximately 2-fold the uptake of 68Ga-DOTA-2P(FAPI)2 (SUVmean, 0.72 ± 0.02 vs. 0.42 ± 0.03, P < 0.001) and more than 4-fold the uptake of 68Ga-FAPI-46 (0.16 ± 0.01, P < 0.001). In the radioligand therapy study, remarkable tumor suppression was observed with the 177Lu-FAPI tetramer in both HT-1080-FAP and U87MG tumor-bearing mice. Conclusion: The satisfactory FAP-binding affinity and specificity, as well as the favorable in vivo pharmacokinetics of the FAPI tetramer, make it a promising radiopharmaceutical for theranostic applications. Improved tumor uptake and prolonged retention of the 177Lu-FAPI tetramer resulted in excellent characteristics for FAPI imaging and radioligand therapy.

Keywords: cancer-associated fibroblasts; dimer; fibroblast activation protein; radioligand therapy; tetramer.

© 2023 by the Society of Nuclear Medicine and Molecular Imaging.

Figures

Graphical abstract
Graphical abstract
FIGURE 1.
FIGURE 1.
Chemical structure of FAPI tetramer DOTA-4P(FAPI)4.
FIGURE 2.
FIGURE 2.
Cell uptake–blocking experiments and competitive cell-binding assay of FAPI-46, DOTA-2P(FAPI)2, and DOTA-4P(FAPI)4 using HT-1080-FAP cells.
FIGURE 3.
FIGURE 3.
(A) Dynamic time–activity curves of 68Ga-DOTA-4P(FAPI)4 in heart, liver, kidney, muscle, and tumor of HT-1080-FAP tumor–bearing mice. (B–D) Representative PET images of 68Ga-DOTA-4P(FAPI)4, 68Ga-DOTA-2P(FAPI)2, and 68Ga-FAPI-46 in HT-1080-FAP tumor–bearing mice.
FIGURE 4.
FIGURE 4.
(A–C) Representative PET images of 68Ga-DOTA-4P(FAPI)4 (bar, SUVmean, 0–1.2) (A), 68Ga-DOTA-2P(FAPI)2 (bar, SUVmean, 0–1.2) (B), and 68Ga-FAPI-46 (bar, SUVmean, 0–0.3 because of low uptake) (C) in U87MG tumor–bearing mice. Arrows point toward tumor. (D) Tumor uptake of 3 radiotracers in U87MG tumors at 0.5–4 h after injection.
FIGURE 5.
FIGURE 5.
Representative PET imaging of 64Cu-NOTA-4P(FAPI)4 and 64Cu-NOTA-2P(FAPI)2 in HT-1080-FAP tumor–bearing mice. Arrows point toward tumor.
FIGURE 6.
FIGURE 6.
Representative SPECT images of 177Lu-DOTA-4P(FAPI)4, 177Lu-DOTA-2P(FAPI)2, and 177Lu-FAPI-46 in HT-1080-FAP tumor–bearing mice. Arrows point toward tumor.
FIGURE 7.
FIGURE 7.
Radioligand therapy with 177Lu-DOTA-4P(FAPI)4, 177Lu-DOTA-2P(FAPI)2, and 177Lu-FAPI-46 in HT-1080-FAP and U87MG tumor–bearing mice. (A) Tumor growth curves and weight changes after treatment in HT-1080-FAP tumors (6/group). (B) Tumor growth curves and weight changes after treatment in U87MG tumors (6/group).

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