Summary

Targeting neoplastic cells has been used as a conventional method for tumor growth and spread determination, however, with the key function of cancer associate fibroblasts (CAFs) being recognized, scientists have been striving to target CAFs for diagnosis and antitumor therapy. CAFs constitute a predominant portion of non-malignant tumor stroma, which in turn account for 90% of the mass in tumors with desmoplastic reactions; and play an essential role in tumor growth, migration and progression. A distinguishing characteristic of CAFs is overexpression of the serine protease fibroblast activation protein (FAP), the presence of which confers a poor prognosis and fast progression of disease in cancer patients. As a result, targeting FAP for both imaging and delivery of the therapeutics has become an attractive approach.
To target FAP for imaging, early attempts were made to develop radiolabeled antibodies. Iodine 131-labeled FAP specific monoclonal antibody F19 (¹³¹I-mAbF19) was evaluated in patients with hepatic metastases from colorectal carcinoma. However, the long circulation time of intact antibodies hampered the delivery of radioactive agents, and suggested that smaller molecules may be suitable for radionuclide imaging. In recent years, the development of a quinoline-based small-molecule radiopharmaceutical based on a FAP-specific inhibitor (FAPI) has revolutionized the strategy for FAP-targeted theranostics. These synthesized FAPIs were able to show specific uptake, rapid internalization, and successful imaging of tumors in vivo and in clinical studies. Nevertheless, due to the enzymatic deiodination with efflux of free iodine of FAPI-01, prolonged incubation period results in lower intracellular radioactivity. To solve this issue, FAPI-02 was synthesized to have the FAP-targeting group chemically linked to the chelator DOTA, resulting in a theranostic compound with favorable pharmacokinetic and biochemical properties. Indeed FAPI-02 acts as an ideal tracer that specifically bind to its target protein FAP to ensure reliable distinction between cancerous and normal tissue as well as achieve a low background signal, resulting in high image contrast. However, with regards to potential tracer's applications for therapeutic purposes, improvement needs to be made to allow: 1) even longer circulation half-life that increases tumor uptakes; 2) slower efflux/longer tumor retention; 3) higher tumor to normal tissue contrast. 
With this in mind, the authors aimed to improve PK and PD profiles of the lead structure FAPI-02, by adding functional chemical groups without significantly affecting the desired biological activity, in order to accomplish a more substantial enhancement in tumor retention while keeping the normal tissue uptake at low level. Albumin serves as a versatile carrier for drug delivery. As the association of bioactive drug to albumin is a reversible action, the albumin-drug complex functions as a drug reservoir that can enhance the drug distribution and bioavailability. Evans blue (EB) is a good example of albumin binding moiety that exhibits relatively high affinity for binding site 1 on serum albumin. Previous studies showed that conjugation of EB derivatives onto targeting molecules remarkably prolongs the blood circulation of drugs, thus improves the therapeutic effectiveness, such as ¹⁷⁷Lu-EB-PSMA for the therapy of castration-resistant prostate cancer; ¹⁷⁷Lu-EB-TATE for the treatment of neuroendocrine tumors; ⁹⁰Y-EB-RGD for the treatment of glioblastoma. Inspired by this improved radiotherapeutic efficacy, this research focuses on investigating whether EB conjugation of FAPI-02 improves the efficacy of FAP-targeting radioligand therapy, especially in regards to tumor retention, tumor-to-normal tissue contrast as well as treatment response.
Glioblastoma multiforme (GBM) is the most aggressive glioma of WHO Grade III and IV. Despite the advances in therapeutics, the prognosis for patients remains poor. FAP was found to be expressed on glioma cells and tumor stroma especially in proximity to blood vessels. Therefore, several studies have been performed to evaluate the PK and biodistribution of ⁶⁸Ga-FAPI-02/04 in glioblastoma patients and achieved the positive results. But up to now, the therapeutic effects of FAPI-02/04 in glioblastoma models have not been researched.
In this study, EB-FAPI-B1, B2, B3 and B4 were designed and synthesized with several major components: a quinoline-based FAPI-02 as lead structure; a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) group that enables radionuclide labeling; poly(ethylene glycol) (PEG, with or without); and truncated EB moiety as an albumin binder. U87MG tumor-bearing mice were selected as the animal model.

Results from nanoScan SPECT/CT

For the imaging studies, about 37 MBq of radiotracers were injected intravenously into female ICR mice and U87MG tumor-bearing mice, whole body SPECT images were acquired at 1, 4, 24, 48, 72 and 96 h p.i. The acquisition parameters were as follows: 56.1, 112.9 and 208.4 keV energy peaks for ¹⁷⁷Lu, window width of 20%, matrix of 256 × 256, medium zoom, and 48 frames. Mice were anaesthetized using 1.5% isoflurane to maintain spontaneous breathing during imaging.
To evaluate the pharmacokinetic characterization of these radiotracers in vivo, SPECT imaging was conducted in healthy ICR mice for different times. As shown in Figure S17, ¹⁷⁷Lu-EB-FAPI-B1, B2, B3, and B4 demonstrated a significant uptake in the blood at 1 h and 4 h post-injection (p.i). The radioactive signal declined in the heart and increased in the kidneys over time. At 24 h and 48 h p.i., the radioactivity in the heart was barely observed while the kidneys possessed a sustained radioactivity. Characteristically, ¹⁷⁷Lu-EB-FAPI-B1 showed low background signals in most organs except for the kidneys, while signal in the kidneys declined at 48 h as compared with that at 24 h p.i. However, higher radioactivities of ¹⁷⁷Lu-EB-FAPI-B2, B3, B4 retained in the liver and kidneys until 48 h p.i. The continuously high radioactivity in the liver and kidneys would result in toxicity to normal organs, which make the three radiotracers less suitable for radioligand therapy in vivo.

Representative whole-body SPECT images of U87MG tumor-bearing mice are shown in Figure 5A and Figure S18A . At 1 h after injection of ¹⁷⁷Lu-EB-FAPI-B1, B2, B3, B4, most of radioactivity accumulated in the heart and blood vessels, and the background signal was also high. At 4 h p.i., obvious tumor uptake was observed for ¹⁷⁷Lu-EB-FAPI-B1, B2, but not for ¹⁷⁷Lu-EB-FAPI-B3, B4. As shown in Figure 5B-C, in the case of ¹⁷⁷Lu-EB-FAPI-B1, high tumor uptake and tumor-to-muscle ratio (T/M: 12.29 ± 0.85) were observed at 24 h p.i., with the peak T/M value occurred at 72 h p.i. (18.67 ± 3.75) and reduced slightly over time (14.15 ± 0.13, 96 h p.i.). In addition, the ratios of tumor-to-heart (T/H: 14.28 ± 2.46), tumor-to-liver (T/L: 3.73 ± 1.21), tumor-to-kidney (T/K: 2.35 ± 0.61) increased more than two-fold from 24 h to 96 h p.i. The low uptakes of nontarget organs led to the high contrast of SPECT images. Compared with ¹⁷⁷Lu-EB-FAPI-B1, the other radiotracers, especially ¹⁷⁷Lu-EB-FAPI-B3 underperformed. It demonstrated higher uptakes in the kidneys (T/K: 0.64 ± 0.04) and liver (T/L: 0.75 ± 0.09), which resulted in low tumor uptake (T/M: 9.97 ± 1.23) at the stated time points (Figure S18B).

• In this research, the authors developed a series of EB conjugated FAPI-02-based radiotracers for tumor theranostics.
• ¹⁷⁷Lu-EB-FAPI-B1 demonstrated greatly improved binding affinity and FAP targeting specificity in vitro. Massively enhanced tumor retention and decreased normal tissue uptake were also observed.
• Most importantly and excitingly, ¹⁷⁷Lu-EB-FAPI-B1 showed remarkable tumor growth suppression effect on U87MG tumor-bearing mice and negligible side effect, suggesting that the EB modified FAPI structures can be applied as promising drugs for radioligand therapy of cancer.

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