Synthesis, Preclinical Evaluation, and a Pilot Clinical PET Imaging Study of 68Ga-Labeled FAPI Dimer

Liang Zhao, Bo Niu, Jianyang Fang, Yizhen Pang, Siyang Li, Chengrong Xie, Long Sun, Xianzhong Zhang, Zhide Guo, Qin Lin, Haojun Chen, Liang Zhao, Bo Niu, Jianyang Fang, Yizhen Pang, Siyang Li, Chengrong Xie, Long Sun, Xianzhong Zhang, Zhide Guo, Qin Lin, Haojun Chen

Abstract

Cancer-associated fibroblasts (CAFs) are crucial components of the tumor microenvironment. Fibroblast activation protein (FAP) is overexpressed in CAFs. FAP-targeted molecular imaging agents, including the FAP inhibitors (FAPIs) 04 and 46, have shown promising results in tumor diagnosis. However, these molecules have a relatively short tumor-retention time for peptide-targeted radionuclide therapy applications. We aimed to design a 68Ga-labeled FAPI dimer, 68Ga-DOTA-2P(FAPI)2, to optimize the pharmacokinetics and evaluate whether this form is more effective than its monomeric analogs. Methods:68Ga-DOTA-2P(FAPI)2 was synthesized on the basis of the quinoline-based FAPI variant (FAPI-46), and its binding properties were assayed in CAFs. Preclinical pharmacokinetics were determined in FAP-positive patient-derived xenografts using small-animal PET and biodistribution experiments. The effective dosimetry of 68Ga-DOTA-2P(FAPI)2 was evaluated in 3 healthy volunteers, and PET/CT imaging of 68Ga-FAPI-46 and 68Ga-DOTA-2P(FAPI)2 was performed on 3 cancer patients. Results:68Ga-DOTA-2P(FAPI)2 was stable in phosphate-buffered saline and fetal bovine serum for 4 h. The FAPI dimer showed high affinity and specificity for FAP in vitro and in vivo. The tumor uptake of 68Ga-DOTA-2P(FAPI)2 was approximately 2-fold stronger than that of 68Ga-FAPI-46 in patient-derived xenografts, whereas healthy organs showed low tracer uptake and fast body clearance. The effective dose of 68Ga-DOTA-2P(FAPI)2 was 1.19E-02 mSv/MBq, calculated using OLINDA. Finally, the PET/CT scans of the 3 cancer patients revealed higher intratumoral uptake of 68Ga-DOTA-2P(FAPI)2 than of 68Ga-FAPI-46 in all tumor lesions (SUVmax, 8.1-39.0 vs. 1.7-24.0, respectively; P < 0.001). Conclusion:68Ga-DOTA-2P(FAPI)2 has increased tumor uptake and retention properties compared with 68Ga-FAPI-46, and it could be a promising tracer for both diagnostic imaging and targeted therapy of malignant tumors with positive expression of FAP.

Keywords: FAPI dimer; PET imaging; cancer-associated fibroblasts; fibroblast activation protein; patient-derived xenografts.

© 2022 by the Society of Nuclear Medicine and Molecular Imaging.

Figures

Graphical abstract
Graphical abstract
FIGURE 1.
FIGURE 1.
Chemical structure of DOTA-2P(FAPI)2.
FIGURE 2.
FIGURE 2.
(A) FAP expression in Huh7 cells and CAFs assayed using Western blotting. (B) Cell uptake assay of 68Ga-DOTA-2P(FAPI)2, 68Ga-FAPI-46, and blocking experiment on CAFs (n = 3). (C) Inhibition of 68Ga-FAPI-46 binding to FAP on CAFs by unlabeled FAPI-46 (2.83 × 10−4 to 10−13 M; n = 3). (D) Inhibition of 68Ga-DOTA-2P(FAPI)2 binding to FAP on CAFs by unlabeled FAPI-46 (1.27 × 10−4 to 10−13 M; n = 3).
FIGURE 3.
FIGURE 3.
Representative static PET imaging of 68Ga-DOTA-2P(FAPI)2 (left top) and 68Ga-FAPI-46 (left bottom) in HCC-PDX-1, and dynamic time–activity curves of 68Ga-DOTA-2P(FAPI)2 (right) in heart, kidney, liver, muscle, and tumor tissues.
FIGURE 4.
FIGURE 4.
(A) Representative static PET imaging of 68Ga-DOTA-2P(FAPI)2 and 68Ga-FAPI-46 in HCC-PDX-2. (B) Representative static PET imaging of 68Ga-DOTA-2P(FAPI)2 in HCC-PDX-1 and HCC-PDX-2 with and without simultaneous injection of unlabeled FAPI-46 as competitor 1 h after administration.
FIGURE 5.
FIGURE 5.
(A) Ex vivo biodistribution of 68Ga-FAPI-46 in HCC-PDX-1, 1 and 4 h after injection (n = 3/group). (B) Ex vivo biodistribution of 68Ga-DOTA-2P(FAPI)2 in HCC-PDX-1, 1 and 4 h after injection, with and without coadministration of unlabeled FAPI-46 as blocking agent (n = 3/group).
FIGURE 6.
FIGURE 6.
68Ga-DOTA-2P(FAPI)2, 10, 30, 60, and 180 min after injection, in healthy volunteers (top), and SUVmean of healthy organs at different time points (bottom).
FIGURE 7.
FIGURE 7.
68Ga-FAPI-46, 1 h after injection, and 68Ga-DOTA-2P(FAPI)2, 1 and 4 h after injection, in patient with metastatic thyroid cancer. Hematoxylin and eosin (H&E) staining and FAP immunohistochemistry staining showed high FAP expression in tumor stroma (×100).

References

    1. Gaggioli C, Hooper S, Hidalgo-Carcedo C, et al. . Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol. 2007;9:1392–1400.
    1. Lakins MA, Ghorani E, Munir H, Martins CP, Shields JD. Cancer-associated fibroblasts induce antigen-specific deletion of CD8 (+) T cells to protect tumour cells. Nat Commun. 2018;9:948.
    1. Loktev A, Lindner T, Burger EM, et al. . Development of fibroblast activation protein-targeted radiotracers with improved tumor retention. J Nucl Med. 2019;60:1421–1429.
    1. Giesel FL, Kratochwil C, Lindner T, et al. . 68Ga-FAPI PET/CT: biodistribution and preliminary dosimetry estimate of 2 DOTA-containing FAP-targeting agents in patients with various cancers. J Nucl Med. 2019;60:386–392.
    1. Chen H, Pang Y, Wu J, et al. . Comparison of [68Ga]Ga-DOTA-FAPI-04 and [18F]FDG PET/CT for the diagnosis of primary and metastatic lesions in patients with various types of cancer. Eur J Nucl Med Mol Imaging. 2020;47:1820–1832.
    1. Ballal S, Yadav MP, Kramer V, et al. . A theranostic approach of [68Ga]Ga-DOTA.SA.FAPi PET/CT-guided [177Lu]Lu-DOTA.SA.FAPi radionuclide therapy in an end-stage breast cancer patient: new frontier in targeted radionuclide therapy. Eur J Nucl Med Mol Imaging. 2021;48:942–944.
    1. Lindner T, Loktev A, Altmann A, et al. . Development of quinoline-based theranostic ligands for the targeting of fibroblast activation protein. J Nucl Med. 2018;59:1415–1422.
    1. Lindner T, Altmann A, Kramer S, et al. . Design and development of 99mTc-labeled FAPI tracers for SPECT imaging and 188Re therapy. J Nucl Med. 2020;61:1507–1513.
    1. Kratochwil C, Giesel FL, Rathke H, et al. . [153Sm]samarium-labeled FAPI-46 radioligand therapy in a patient with lung metastases of a sarcoma. Eur J Nucl Med Mol Imaging. 2021;48:3011–3013.
    1. Watabe T, Liu Y, Kaneda-Nakashima K, et al. . Theranostics targeting fibroblast activation protein in the tumor stroma: 64Cu- and 225Ac-labeled FAPI-04 in pancreatic cancer xenograft mouse models. J Nucl Med. 2020;61:563–569.
    1. Loktev A, Lindner T, Mier W, et al. . A tumor-imaging method targeting cancer-associated fibroblasts. J Nucl Med. 2018;59:1423–1429.
    1. Assadi M, Rekabpour SJ, Jafari E, et al. . Feasibility and therapeutic potential of 177Lu-fibroblast activation protein inhibitor-46 for patients with relapsed or refractory cancers: a preliminary study. Clin Nucl Med. 2021;46:e523–e530.
    1. Baum RP, Schuchardt C, Singh A, et al. . Feasibility, biodistribution and preliminary dosimetry in peptide-targeted radionuclide therapy (PTRT) of diverse adenocarcinomas using 177Lu-FAP-2286: first-in-human results. J Nucl Med. June 24, 2021. [Epub ahead of print].
    1. Toms J, Kogler J, Maschauer S, et al. . Targeting fibroblast activation protein: radiosynthesis and preclinical evaluation of an 18F-labeled FAP inhibitor. J Nucl Med. 2020;61:1806–1813.
    1. Hidalgo M, Amant F, Biankin AV, et al. . Patient-derived xenograft models: an emerging platform for translational cancer research. Cancer Discov. 2014;4:998–1013.
    1. Lang L, Li W, Guo N, et al. . Comparison study of [18F]FAl-NOTA-PRGD2, [18F]FPPRGD2, and [68Ga]Ga-NOTA-PRGD2 for PET imaging of U87MG tumors in mice. Bioconjug Chem. 2011;22:2415–2422.
    1. Li ZB, Cai W, Cao Q, et al. . 64Cu-labeled tetrameric and octameric RGD peptides for small-animal PET of tumor alphavbeta3 integrin expression. J Nucl Med. 2007;48:1162–1171.
    1. Wu Z, Li ZB, Chen K, et al. . MicroPET of tumor integrin alphavbeta3 expression using 18F-labeled PEGylated tetrameric RGD peptide (18F-FPRGD4). J Nucl Med. 2007;48:1536–1544.
    1. Zhao L, Chen H, Guo Z, et al. . Targeted radionuclide therapy in patient-derived xenografts using 177Lu-EB-RGD. Mol Cancer Ther. 2020;19:2034–2043.
    1. Chen H, Zhao L, Fu K, et al. . Integrin alphavbeta3-targeted radionuclide therapy combined with immune checkpoint blockade immunotherapy synergistically enhances anti-tumor efficacy. Theranostics. 2019;9:7948–7960.
    1. Chen H, Zhao L, Ruan D, et al. Usefulness of [68Ga]Ga-DOTA-FAPI-04 PET/CT in patients presenting with inconclusive [18F]FDG PET/CT findings. Eur J Nucl Med Mol Imaging. 2021;48:73–86.
    1. Stabin MG, Sparks RB, Crowe E. OLINDA/EXM: the second-generation personal computer software for internal dose assessment in nuclear medicine. J Nucl Med. 2005;46:1023–1027.
    1. Meyer C, Dahlbom M, Lindner T, et al. . Radiation dosimetry and biodistribution of 68Ga-FAPI-46 PET imaging in cancer patients. J Nucl Med. 2020;61:1171–1177.
    1. Jokar N, Velikyan I, Ahmadzadehfar H, et al. . Theranostic approach in breast cancer: a treasured tailor for future oncology. Clin Nucl Med. 2021;46:e410–e420.
    1. Wang S, Zhou X, Xu X, et al. . Clinical translational evaluation of Al18F-NOTA-FAPI for fibroblast activation protein-targeted tumour imaging. Eur J Nucl Med Mol Imaging. 2021;48:4259–4271.
    1. Liu S. Radiolabeled cyclic RGD peptides as integrin alphavbeta3-targeted radiotracers: maximizing binding affinity via bivalency. Bioconjug Chem. 2009;20:2199–2213.
    1. Li ZB, Chen K, Chen X. 68Ga-labeled multimeric RGD peptides for microPET imaging of integrin alphavbeta3 expression. Eur J Nucl Med Mol Imaging. 2008;35:1100–1108.

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