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Preclinical evaluation and first-in-human study of [18F]AlF-FAP-NUR for PET imaging cancer-associated fibroblasts

EJNMMI Research volume 14, Article number: 87 (2024) Cite this article

Abstract

Background

Fibroblast activation protein (FAP) has gained attention as a promising molecular target with potential utility for cancer diagnosis and therapy. [68Ga]Ga-labeled FAP-targeting peptides have been successfully applied to positron emission tomography (PET) imaging of various tumor types. To meet the applicable demand for peptide-based FAP tracers with high patient throughput, we herein report the radiosynthesis, preclinical evaluation, and the first-in-human imaging of a novel [18F]F-labeled FAP-targeting peptide.

Results

[18F]AlF-FAP-NUR was automatedly prepared within 45 min with a non-decay corrected radiochemical yield of 18.73 ± 4.25% (n = 3). Compared to [68Ga]Ga-FAP-2286, the [18F]F-labeled peptide demonstrated more rapid, higher levels of cellular uptake and internalization, and lower levels of cellular efflux in HT1080-FAP cells. Micro-PET imaging and biodistribution studies conducted on xenograft mice models revealed a similar distribution pattern between the two tracers. However, [18F]AlF-FAP-NUR demonstrated significantly higher tumor-specific uptake resulting in improved Tumor-Background Ratios (TBRs). In the patients, a significant accumulation of [18F]AlF-FAP-NUR was found in the primary tumor. High uptake of the tracer within the bladder indicated that its major route of excretion was through urine.

Conclusions

Based on the physical imaging properties and longer half-life of [18F]F, [18F]AlF-FAP-NUR exhibited promising characteristics such as enhanced tumor-specific accumulation and elevated TBRs, which made it a viable candidate for further clinical investigation.

Trial registration

www.Chictr.org.cn, ChiCTR2300076976 Retrospectively registered 25 October 2023. at, URL: https://www.chictr.org.cn/showproj.html?proj=206753.

Introduction

Fibroblast activation protein (FAP) is specifically upregulated on the cell surface of cancer-associated fibroblasts (CAFs) but has little or no expression in quiescent fibroblasts within normal adult tissues [1,2,3,4,5]. The differential expression pattern highlights the important role of FAP as a potential target for cancer imaging and therapy [6,7,8,9,10]. The quinoline-based small molecule inhibitors targeting FAP, known as FAPIs (including FAPI-04 [6, 11], FAPI-46 [12, 13], and FAPI-74 [14, 15]), labeled with [68Ga]Ga for use in positron emission tomography (PET) scans, have demonstrated promising results in the diagnosis of various types of cancers [15,16,17]. Furthermore, the recent study on the SuFEx strategy has demonstrated a significant improvement in diagnostic sensitivity and therapeutic efficacy by modifying FAPI-04 as a covalent targeted radioligand (CTR), leading to enhanced tumor retention and minimized off-target effects on healthy tissues [18]. These small molecule tracers exhibit a high affinity for FAP and show enhanced selectivity towards tumors compared to normal tissues, leading to preferential accumulation within tumors and enabling more precise detection of cancerous lesions.

Additionally, a novel class of modalities has emerged that employs cyclic peptides as alternative binding motifs to FAP. Notably, within this category, FAP-2286 is a cyclic peptide that exhibits specific and selective binding to FAP, resulting in robust tumor accumulation [8,9,10, 17, 19]. PET imaging using [68Ga]Ga-FAP-2286 produced high-quality diagnostic scans that could significantly facilitate the detection of metastatic disease in patients with solid tumors [6, 8,9,10]. The clinical outcomes observed with [68Ga]Ga-FAP-2286 are comparable to those seen with [68Ga]Ga-FAPI-46 in oncology, highlighting its high sensitivity and specificity in detecting primary tumors as well as metastatic lesions [17]. Moreover, ongoing studies have preliminarily demonstrated favorable pharmacokinetic and pharmacological characteristics of [68Ga]Ga-3BP-3940 [20] (a urea-modified derivative of FAP-2286), including low renal and hepatic uptake along with a high TBRs.

Despite these promising preliminary findings achieved with [68Ga]Ga-labeled FAP radiotracers, the limited nuclide availability and economic issues related to 68Ge/68Ga generators limit their use in clinical practice and drive the pursuit for alternative radioisotopes. [18F]F, possessing an appropriately extended half-life (T1/2 ≈ 109.8 min, 96.7% β+), is widely utilized in the development of PET imaging agents and can be produced in significantly larger quantities using a medical cyclotron. In comparison to [68Ga]Ga, [18F]F exhibits a shorter positron range and higher positron yield, thereby offering superior spatial resolution and enhanced contrast. Considering the advantageous physical properties of [18F]F, our previous study developed a novel cyclic peptide molecule, [18F]AlF-FAP-2286, which replaced DOTA with NOTA based on the structure of FAP-2286 (Fig. 1) [21]. [18F]AlF-FAP-2286 outperformed [18F]FDG in imaging gastric cancer and some metastatic lesions. However, it exhibited significant physiological uptake and was primarily excreted through the hepatobiliary system, posing challenges in detecting lesions within the liver, gallbladder, and intestines.

Fig. 1
figure 1

The chemical structures of [68Ga]Ga-FAP-2286, [18F]AlF-FAP-2286 and [18F]AlF-FAP-NUR

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The incorporation of the urea moiety in peptides has demonstrated its advantageous role as a substitute for amide bonds, contributing to desirable pharmacological properties such as enhanced protease resistance and increased affinity towards target proteins [22, 23]. Simultaneously, the urea moiety offers opportunities for attaching other functional groups to finely adjust the hydrophobic-hydrophilic balance and locally modify conformation. To enhance urinary system excretion and reduce physiologic intestinal system uptake, the present study aims to develop an [18F]F-labeled cyclic peptide targeting FAP ([18F]AlF-FAP-NUR) with a urea-modified tail at the N-terminus. Additionally, this manuscript presents the radiosynthesis, preclinical evaluation, and first-in-human imaging of this innovative cyclic peptide.

Materials and methods

Radiosynthesis

NOTA-FAP-NUR (Nanchang Tanzhen Biotechnology Co., Ltd., Jiangxi, China) (Fig. 1) was radiolabeled with [18F]F. For [18F]AlF-labelings, the tracer was synthesized on a modified GE TRACERlab FxFN platform according to the steps shown in Supplementary Fig. S1. The irradiated [18O]H2O was bypassed through the Sep-Pak QMA Plus Light Cartridge (preconditioned with 10 mL ethanol and 10 mL water) into the vessel for [18O]H2O recovery in vacuo. Subsequently, the [18F]F− (22–29 GBq) trapped on the QMA cartridge was eluted with 0.5 mL of 0.9% NaCl aqueous from Vial 1 into the reaction vessel. Drying was accomplished by azeotropic distillation using 1 mL of acetonitrile from Vial 2. After completing the drying process, a mixture solution containing 400 µL acetonitrile, 250 µL 0.2 M NaOAc buffer (pH 4.0), 7.0 µL 10.0 mM AlCl3 in 0.2 M NaOAc buffer (pH 4.0), and 150 µL precursor (1 mg/mL) in 0.125 M ascorbic acid was added to the reaction vessel from Vial 3. After being heated for 15 min at 100 °C, the mixture was cooled to 70 °C and diluted with 10 mL of water from Vial 5. Next, the dilute solution was transferred over a C18 Light Cartridge (preconditioned with 10 mL ethanol and 10 mL water). Afterward, the cartridge was washed sequentially with 10 mL of water from Vial 6 and 5 mL of water from Vial 8. The product was eluted with 1.5 mL 50% EtOH solution from Vial 7 into the pre-loaded collection vial (6.5 mL 0.9% sterile NaCl for injection containing 10 mg/mL ascorbic acid). The final product solution of 8 mL was transferred into the sterile vial by sterilizing filtration using a Millex GV 33 mm 0.22 μm filter. In addition, a [68Ge]Ge/[68Ga]Ga generator obtained from iThemba LABS was employed for the labeling of [68Ga]Ga. The radiosynthesis of [68Ga]Ga-FAP-2286 followed the previously described method [10].

Stability and partition coefficient

[18F]AlF-FAP-NUR (100 µCi, 10 µL) was added to saline (90 µL) and the mixture was incubated at 37 ℃ for 4 h. The mixture was injected directly into the radio-HPLC (Thermo Fisher Scientific, USA) for analysis. The radio-chemical purity was automatically analyzed by the radio-HPLC. Detailed information is provided in the Supplementary data.

The tracers were added to equal volumes of n-octanol and phosphate-buffered saline (PBS) buffer (0.01 M, pH = 7.4), and centrifuged at 12,000 × rpm for 5 min (n = 3). Then, the mixture was centrifuged, and equal volumes of n-octanol and PBS solutions were taken separately to be counted by an automatic γ-counter (Hidex AMG, Hidex Oy, Finland), and the average LogP value was calculated.

Cell culture and preparation of xenograft models

293T, A549, HT1080 cell lines and the cells transfected with human FAP genes, including 293T-FAP, A549-FAP, and HT1080-FAP were cultured in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum (Bovogen, Australia) and 1% penicillin-streptomycin solution (Cytiva, USA) at 37 ℃/5% carbon dioxide.

All animal experiments were conducted in compliance with a protocol approved by the Guangzhou Medical University Institutional Animal Care and Use Committee (No. 20230330). Normal male Kunming (KM) mice (6 weeks old with body weight of 25–34 g) were purchased from Zhuhai BesTest Bio-Tech Co., Ltd., and male BALB/c-nude mice (4–5 weeks old with body weight of 16–22 g) were purchased from GemPharmatech Co., Ltd. Mice were housed under standard conditions with temperature and light control (12-h-light/12-h-dark cycle) and had free access to water and food. For the tumor models, 293T-FAP or 293T cells (5 × 106/mouse, n = 8 mice/group) were subcutaneously injected into the right flank of male BALB/c-nude mice, respectively.

Cell assays

For cell uptake experiments, HT1080-FAP cells (5 × 104/well) or HT1080 (5 × 104/well) were seeded in 24-well plates for 24 h and incubated at 37 °C with radiolabeled tracers (74 kBq/well) in 1 mL of serum-free medium for indicated times. Non-specific binding was determined by co-incubating with DOTA-FAP-2286 (3 µM/well). For internalization experiments, the radiotracer-incubated cells were washed twice with PBS (pH 7.4), followed by glycine-HCl (0.05 M, pH = 2.8) solution to distinguish between cell surface-bound (acid-releasable) and internalized (acid-resistant) radioligand. For efflux experiments, cells were preincubated with [18F]AlF-FAP-NUR or [68Ga]Ga-FAP-2286 for 1 h, and subsequently incubated at 37 °C in a medium free of radiotracers and serum for different time points. At the same time, a group without adding medium was kept measuring the total amount bound to the cells. Then the medium was removed, and the cells were washed twice with cold PBS and subsequently lysed with 0.2 mL of NaOH (1 M). Cell lysates were collected, and the radioactivity was determined using a γ-counter. The efflux rate was calculated by using the radioactivity of the total bound to the cells (0 min) as the denominator and the radioactivity at different time points as the numerator.

For competitive FAP binding assays, 293T-FAP cells were incubated with [177Lu]Lu-FAP-2286 (74 kBq/well) in the presence of different concentrations (10− 5–10− 11 M) of competing non-radioactive ligands for 60 min. The cell incubation process was performed by Brandel M-48T Cell Harvester (Biomedical Research & Development Laboratories, Inc., Gaithersburg, MD, USA). Then the cells were collected, and the radioactivity was measured with a γ-counter.

Micro PET/CT imaging and ex vivo biodistribution in mice

Micro PET/CT imaging and biodistribution studies were conducted when the tumor diameter reached 5–8 mm. PET/CT imaging was performed using a PET scanner (MadicLab PSA071, Shandong Madic Technology Co., Ltd., China). Micro-PET scanning was performed after intravenous injection of [68Ga]Ga-FAP-2286 (0.1 mL, 7.4 MBq) or [18F]AlF-FAP-NUR (0.1 mL, 7.4 MBq). The acquisition time of PET scanning for FAP-positive tumor-bearing mice was 120 min, then it was framed at a rate of 5 min per frame. The FAP-negative tumor-bearing mice had a 10-min static scan after receiving the equivalent medication injection for 60 min. Before static scan, mice were able to move freely. The CT scan parameters were tube voltage of 80 kV, tube current of 0.7 mA, and voxel spacing of 200 μm. The PET image was used to determine the radiotracer uptake, and the CT image was used for the attenuation correction of PET images and localization of radiotracer uptake sites. The images and volumes of interest (VOI) were produced using PMOD software (version 4.4, PMOD Technologies LLC, Switzerland).

To characterize the normal biodistribution of [18F]AlF-FAP-NUR in normal male KM mice, mice were divided into 5 groups (n = 4/group) and injected intravenously with 100 µL of [18F]AlF-FAP-NUR (2.22–3.70 MBq/mouse). At different time points after injection, the mice were sacrificed, and blood samples and organs of interest were collected, weighed and counted with a γ-counter. Meanwhile, to further verify the biodistribution in xenograft mice, male BALB/c-nude mice bearing 293T-FAP tumors were divided into two groups (n = 4/group) and injected intravenously with 100 µL of [68Ga]Ga-FAP-2286 or [18F]AlF-FAP-NUR (2.22–3.70 MBq/mouse), respectively. At 1 h after injection, they were subjected to the same operation as described above.

Patient enrolment

The clinical translational study of [18F]AlF-FAP-NUR was approved by the institutional review board of the First Affiliated Hospital of Guangzhou Medical University (ES-2023-083-01) and was registered on the www.Chictr.org.cn under the identification number ChiCTR2300076976. Oral and written informed consent was obtained from all participants. The inclusion criteria were as follows: (1) adult participants (aged 18 years or older), (2) patients with suspected tumors who couldn’t be clearly diagnosed as tumors using other imaging methods such as ultrasound, CT and MRI, and (3) patient’s general condition was acceptable and could tolerate PET examination. Exclusion criteria were as follows: (1) non-tumor disease, and (2) patients who were pregnant.

Clinical PET/CT imaging

For the clinical translational study of [18F]AlF-FAP-NUR, we enrolled two patients, including a 65-year-old woman with a preliminary diagnosis of breast cancer through CT scan, and a 70-year-old woman with a right upper lung lesion. Both patients received an intravenous injection of [18F]AlF-FAP-NUR at a radioactivity dose of 5.55 MBq/kg (0.15 mCi/kg). Approximately 50 min after intravenous administration, static PET/CT scans were acquired using uMI Panorama system (United Imaging Healthcare, Shanghai, China) from the top of the skull to the middle of the femur. The PET scan parameters were: 1 min/bed position, 4 positions in total, 256 × 256 matrix. The CT scan parameters were: 120 kV, auto-mAs, 512 × 512 matrix, 0.5 s per tube rotation, slice thickness of 3.75 mm, and pitch of 0.9625. All data were reconstructed on the workstation (uWS-MI R002.5.0). Reconstruction for all imaging data was performed with an ordered subset expectation maximization algorithm (OSEM) with three iterations per ten subsets. For standardized uptake value (SUV) calculations, a sphere with proper diameter was placed inside the organ parenchyma for evaluating the maximum SUV (SUVmax) of tumors and mean SUV (SUVmean) of normal organs, then the TBRs were calculated as the ratio tumor-SUVmax/normal-organ-SUVmean. Two experienced nuclear medicine physicians independently reviewed all images, and any discordant results were resolved by consensus.

Data analysis and statistics

Statistical analysis was performed using the SPSS 27.0 software (IBM Corp., Armonk, NY, USA) and GraphPad Prism 9.0 (GraphPad Software, Boston, MA, USA). Data were presented as mean ± SD. An unpaired t-test was used to evaluate the differences in each organ uptake between [18F]AlF-FAP-NUR and [68Ga]Ga-FAP-2286 PET/CT. Statistical significance was defined as P < 0.05.

Results

Radiolabeling, in vitro stability and partition coefficient

[18F]F-labeled FAP peptide was successfully automated preparation via [18F]AlF-chelation on the GE tracerlab FXFN module, resulting in moderate yields of 18.73 ± 4.25% (n = 3, non-decay corrected) with a specific activity of 48.28 ± 18.14 GBq/µmol at the end of synthesis (EOS). The radiochemical purity (RCP) exceeded 95%, which was determined by radio high-performance liquid chromatogram (HPLC). The total synthesis time, starting from the end of [18F]F transfer to the synthesis module, for obtaining the purified product [18F]AlF-FAP-NUR was within 45 ± 2 min. The partition coefficient (LogP) of [18F]AlF-FAP-NUR and [68Ga]Ga-FAP-2286 were −2.45 ± 0.07 (n = 3) and −2.61 ± 0.01 (n = 3), respectively (Supplementary Table S1). The stability of [18F]AlF-FAP-NUR remained above 90% after 4 h in vitro (Fig. 2A). In addition, the preparation of [68Ga]Ga/[177Lu]Lu-FAP-2286 was conducted in accordance with the previously published protocol [10].

Fig. 2
figure 2

In vitro stability and in vitro experiment results of [68Ga]Ga-FAP-2286 and [18F]AlF-FAP-NUR. (A) In vitro stability of [18F]AlF-FAP-NUR at 0.5, 1, 2, and 4 h. (B) The binding affinity of DOTA-FAP-2286 and NOTA-FAP-NUR in 293T-FAP cells. (C) Uptake in HT1080-FAP cells and HT1080 cells after 60 min of incubation, with and without blocking using DOTA-FAP-2286 as a competitor. (D) Uptake in HT1080-FAP cells after incubation for 5–120 min. (E) Internalization into HT1080-FAP cells after incubation for 5–120 min. (F) Efflux kinetics after 60 min of incubation of HT1080-FAP cells with radiolabeled compounds, followed by incubation with compound-free medium for 5–120 min. Data of the binding affinity were represented as mean ± SD (n = 3/group), while the others’ consist of n = 4/group. ****, P < 0.0001

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Cell assays

NOTA-FAP-NUR exhibited significantly higher binding affinity compared to DOTA-FAP-2286, as evidenced by the IC50 (31.7 nM vs. 202.9 nM) shown in Fig. 2B. The cell uptake of [18F]AlF-FAP-NUR in HT1080-FAP cells exhibited a higher level compared to that of [68Ga]Ga-FAP-2286 (60 min: 56.92 ± 1.98% ID/million cells vs. 46.29 ± 6.84% ID/million cells, 120 min: 65.77% ± 0.78% ID/million cells vs. 42.47% ± 4.38% ID/million cells) (Fig. 2D). Additionally, the cell uptake could be significantly blocked by DOTA-FAP-2286, approaching the levels observed in FAP-negative cells (HT1080-FAP: 120 min: 0.75 ± 0.10% ID/million cells vs. 4.61 ± 1.23% ID/million cells) (Fig. 2C). The internalization experiments demonstrated a rapid and high uptake of [18F]AlF-FAP-NUR and [68Ga]Ga-FAP-2286 after 60 min of incubation with 61% ID/million cells and 32% ID/million cells internalized activity, respectively (Fig. 2E). [18F]AlF-FAP-NUR displayed slower excretion than [68Ga]Ga-FAP-2286 (120 min: < 52% vs. < 75%), according to the results of the efflux experiments (Fig. 2F). The cell assays of 293T-FAP cells were shown in Supplementary Fig. S2.

Ex vivo biodistribution

According to the normal male KM mice’s biodistribution data, the uptake of [18F]AlF-FAP-NUR gradually decreased in most organs over time (Fig. 3A). The tracer was primarily excreted through the kidneys and exhibited a substantial decrease within a 30-min timeframe (5 min vs. 30 min: 18.33 ± 1.75% ID/g vs. 5.58 ± 0.89% ID/g). The biodistribution data of 293T-FAP tumor-bearing mice (Fig. 3B) revealed that, at 1 h post-injection, the uptake of [18F]AlF-FAP-NUR by tumors was two-fold higher compared to that of [68Ga]Ga-FAP-2286 (26.99 ± 10.10% ID/g vs. 11.55 ± 3.11% ID/g, respectively; P = 0.031). The TBRs of [18F]AlF-FAP-NUR, except for the tumor-bone ratio (Fig. 3C), exhibited higher values compared to those of [68Ga]Ga-FAP-2286, particularly in terms of the tumor-muscle ratio (61.31 ± 18.53 vs. 22.66 ± 8.08, respectively; P = 0.021) and tumor-kidney ratio (5.09 ± 2.60 vs. 1.63 ± 0.58, respectively; P = 0.041).

Fig. 3
figure 3

In vivo biodistribution results. (A) Biodistribution results of [18F]AlF-FAP-NUR in normal male KM mice after intravenous injection for 5–120 min. (B) Biodistribution studies of [18F]AlF-FAP-NUR and [68Ga]Ga-FAP-2286 in male 293T-FAP tumor-bearing mice 60 min after intravenous injection. (C) TBRs of two radiotracers in male 293T-FAP tumor-bearing mice at 60 min. All the data are expressed as mean ± SD (n = 4). *, P < 0.05

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Micro-PET imaging

Fused PET and CT images are shown in Fig. 4A-D. [18F]AlF-FAP-NUR exhibited a higher level of tumor uptake compared to [68Ga]Ga-FAP-2286, particularly in 293T-FAP tumors (at 60 min: 17.85 ± 5.12% ID/g vs. 7.62 ± 1.83% ID/g, respectively) (Fig. 4A and C). Rapid clearance of [18F]AlF-FAP-NUR was observed in the kidneys of 293T-FAP tumor-bearing mice, with a reduction of approximately 50% at 45 min p.i. Notably, [18F]AlF-FAP-NUR demonstrated higher TBRs in 293T-FAP tumor-bearing mice compared to [68Ga]Ga-FAP-2286 at 60 min p.i., with a particularly significant difference observed in the tumor-muscle ratio (55.13 vs. 13.60, P < 0.05) (Fig. 4E). Furthermore, [18F]AlF-FAP-NUR exhibited low physiologic accumulation in non-targeted organs, including muscle tissue (at 60 min: 0.35 ± 0.13% ID/g) (Fig. 4A and C). In A549-FAP and A549 tumor-bearing mice, similar results could be observed. Compared with [68Ga]Ga-FAP-2286, [18F]AlF-FAP-NUR had a higher level of tumor accumulation (at 60 min: 6.67 ± 2.96% ID/g vs. 3.37 ± 0.79% ID/g, respectively) (Fig. 4B and D). Especially in the Tumor/Muscle Ratio, the TBRs of [18F]AlF-FAP-NUR were slightly higher than that of [68Ga]Ga-FAP-2286 (8.44 vs. 6.72, P = 0.492) (Fig. 4F). Based on the H&E staining and immunohistochemistry (IHC) results shown in Supplementary Fig. S3, it is evident that FAP exhibits significant overexpression in FAP-positive tumors, whereas FAP expression is virtually absent in FAP-negative tumors.

Fig. 4
figure 4

Micro-PET images and time-activity curves of [68Ga]Ga-FAP-2286 and [18F]AlF-FAP-NUR in male tumor-bearing mice. (A, C) Uptake of [68Ga]Ga-FAP-2286 and [18F]AlF-FAP-NUR was assessed at 60 min in 293T-FAP and 293T tumor-bearing mice (n = 4/group). (B, D) Uptake of [68Ga]Ga-FAP-2286 and [18F]AlF-FAP-NUR was assessed at 60 min in 293T-FAP and 293T tumor-bearing mice (n = 4/group). Time-activity curves in A-D show the accumulation of radiotracers after injection for 5–120 min in different organs. (E) TBRs of two radiotracers in different xenografts at 60 min, including 293T-FAP and 293T tumor-bearing mice. (F) TBRs of two radiotracers in different xenografts at 60 min, including A549-FAP and A549 tumor-bearing mice. All the data are expressed as mean % ID/g ± SD (n = 4)

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Clinical imaging

The results were shown in Fig. 5. Informed consent was obtained from the subjects prior to conducting the [18F]AlF-FAP-NUR PET/CT scans. Two patients were evaluated in this study, both of whom were confirmed by histopathology (Fig. 5). The primary tumors exhibited intense radiotracer uptake in [18F]AlF-FAP-NUR PET/CT imaging (patient 1: SUVmax = 21.20 g/mL; patient 2: SUVmax = 14.19 g/mL). The muscle uptake was relatively low (patient 1: SUVmean = 0.65 g/mL; patient 2: SUVmean = 1.32 g/mL). As a result, high TBRs could be obtained from the images (TBR = 32.62 for patient 1, and TBR = 10.75 for patient 2). Furthermore, FAP expression was detected in their malignant lesions according to the IHC results. Pathological findings revealed that the right breast of a 65-year-old woman had ductal carcinoma while a 70-year-old woman had invasive adenocarcinoma of the upper right lung.

Fig. 5
figure 5

The clinical [18F]AlF-FAP-NUR PET/CT images, H&E results and FAP-IHC staining of primary lesions in patients. (A) A patient with right breast ductal cancer. (B) A patient with infiltrating adenocarcinoma of the right lung. Scale, 50 μm

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Discussion

The expression level of FAP is associated with various aspects of tumor biology, including growth, invasion, metastasis, and therapy response. PET imaging utilizing [68Ga]Ga-FAP-2286 enables non-invasive detection of FAP expression levels in tumors, thereby assisting in early diagnosis, treatment planning, and therapy response monitoring [6, 10, 17]. However, implementing [68Ga]Ga-FAP-2286 PET imaging in large medical centers with high patient throughput faces challenges. One challenge is the limited production capacity of the [68Ge]Ge/[68Ga]Ga generator used to produce the radiotracers. Intensive usage may exceed supply at times, causing delays or limitations in performing PET scans. Another challenge is the short half-life (68 min) of the [68Ga]Ga nuclide itself. Efficient coordination between production and administration is necessary to ensure timely delivery to patients, especially when multiple patients require sequential scans within a limited timeframe. To address the aforementioned issues, we hereby developed a novel [18F]F-labeled FAP-targeting peptide, [18F]AlF-FAP-NUR, for PET imaging to assess its specific targeting towards FAP in preclinical experiments. Additionally, we conducted a first-in-human study in two patients with breast cancer and lung cancer, respectively.

In cell-based binding affinity assays, NOTA-FAP-NUR exhibited higher binding affinity to FAP with an IC50 of 31.7 nM compared to DOTA-FAP-2286 with an IC50 of 202.9 nM. Notably, the binding affinity of NOTA-FAP-NUR was approximately seven-fold that of the known tracer DOTA-FAP-2286, implying that the introduction of a urea-modified tail at the N-terminus could enhance its affinity towards FAP. The cellular uptake and internalization rate of [18F]AlF-FAP-NUR and [68Ga]Ga-FAP-2286 gradually raised over time within 2-hour incubation, but higher values of [18F]AlF-FAP-NUR were obtained. Furthermore, the efflux experiments demonstrated a significantly higher retention rate of radioactivity for [18F]AlF-FAP-NUR (60%) after a 2-hour incubation compared to only 30% retained by [68Ga]Ga-FAP-2286, indicating prolonged cellular retention of [18F]AlF-FAP-NUR. Importantly, DOTA-FAP-2286 could effectively inhibit the accumulation of [18F]AlF-FAP-NUR in the FAP-positive cells to a level of uptake comparable to that observed in the FAP-negative cells, thus confirming the specific binding between the [18F]F-labeled peptide and FAP.

To investigate the biodistribution and clearance properties of the [18F]F-labeled peptide, we conducted dissection studies on normal KM mice (n = 4/group). The tracer showed rapid in vivo clearance through the kidneys, with extremely low uptake in most normal organs, including the brain, muscle, stomach, heart, lung, blood, pancreas, and liver. The uptake values of [18F]AlF-FAP-NUR in the kidneys at 5 min were about 20% ID/g and decreased to only 2% ID/g after 120 min. Therefore, our biodistribution studies confirmed that the primary excretion route for this tracer is through the kidneys. Importantly, no abnormal accumulation of [18F]AlF-FAP-NUR was found in the mice bone suggesting an absence of defluorination or bone marrow uptake. It is noteworthy that this finding demonstrated an obvious distinction in comparison to the previously reported [18F]F-labeled small molecular FAPI tracers, such as [18F]AlF-FAPI-42 [24,25,26], [18F]FGlc-FAPI [27], [18F]AlF-FAPI-74 [14, 28], and [18F]AlF-P-FAPI [19], suggesting enhanced in vivo stability or an alternative metabolic pathway for [18F]AlF-FAP-NUR. Additionally, to facilitate a comparison of tumor uptake between [18F]AlF-FAP-NUR and [68Ga]Ga-FAP-2286, biodistribution studies were also conducted on 293T-FAP subcutaneous xenograft mice (Fig. 3). Although both ligands exhibited a comparable biodistribution pattern at 60 min p.i., [18F]AlF-FAP-NUR demonstrated an approximately 2.5-fold higher tumor accumulation, leading to higher tumor-to-nontarget tissue ratios.

The encouraging results obtained from in vitro cellular investigations and in vivo biodistribution studies, including the demonstration of high affinity towards FAP, specific tumor-targeting properties, and favorable pharmacokinetic characteristics, have motivated us to further explore the potential of this [18F]F-labeled peptide as a FAP-targeting tracer for PET imaging of tumors. To further investigate the pharmacokinetics and FAP specificity, we conducted a comparative PET/CT imaging study using [18F]AlF-FAP-NUR and [68Ga]Ga-FAP-2286 in mice models bearing 293T, A549, A549-FAP and 293T-FAP tumors, respectively (Fig. 4). In FAP-positive tumors such as 293T-FAP and A549-FAP, the average uptake value of [18F]AlF-FAP-NUR was found to be significantly higher compared to that of [68Ga]Ga-FAP-2286 in the same tumor-bearing mice model. The accumulation of [18F]AlF-FAP-NUR and [68Ga]Ga-FAP-2286 was significantly greater in the 293T-FAP tumors compared to the A549-FAP tumors, but no uptake was detected in the FAP-negative tumors, which aligns with the results obtained from the IHC of FAP analysis (Supplementary Fig. S3). The time-activity curves (Fig. 4A and D) exhibited rapid accumulation of both radiotracers in 293T-FAP tumors, with a subsequent plateau in tumor uptake observed from 60 min p.i. to 120 min p.i., indicating favorable tumor retention. Furthermore, the kidney clearance behavior of [18F]AlF-FAP-NUR is also reflected by the respective maximum intensity projections depicted in Fig. 4A and D consistent with the findings from the biodistribution studies. Collectively, the preclinical PET data indicate that [18F]AlF-FAP-NUR exhibited enhanced tumor uptake and superior TBRs compared to the first-generation peptide [68Ga]Ga-FAP-2286, suggesting its potential as a candidate for PET imaging of FAP. Compared to our previous study, both [18F]AlF-FAP-NUR and [18F]AlF-FAP-2286 exhibit comparable tumor uptake in 293T-FAP xenograft mice, however, [18F]AlF-FAP-NUR demonstrates predominant renal excretion [21].

Based on the preclinical investigations, we further translated the findings from animal models into pilot PET/CT studies in two patients with breast cancer and lung cancer, respectively, to explore the potential clinical use of the [18F]F-labeled FAP-targeting peptide. The PET/CT scan results of the tracer exhibited a pronounced uptake in tumor lesions while demonstrating relatively limited accumulation in most normal tissues, thereby yielding a high TBR. No accumulation of [18F]AlF-FAP-NUR was observed in the bones of human subjects during the scans, indicating the absence of defluorination and aligning with the tracer’s findings in mice. Additionally, notable gallbladder retention of [18F]AlF-FAP-NUR was observed in humans, which has not been noticed in the previous studies of [68Ga]Ga-FAP-2286 [6, 8,9,10, 12, 17] but shows a resemblance to the [18F]F-labeled small molecular FAPI tracers, such as [18F]AlF-FAPI-42 [16, 24,25,26], and [18F]AlF-FAPI-74 [14, 16, 28]. This may be ascribed to the higher lipophilicity of [18F]AlF-FAP-NUR, since the LogP of [18F]AlF-FAP-NUR was calculated to be –2.45 ± 0.07 (n = 3), higher than that of [68Ga]Ga-FAP-2286 (–2.61 ± 0.01, n = 3). Our previous study on [18F]AlF-FAP-2286 demonstrated that the increased excretion in the liver and gallbladder after 45 min post-injection, along with a relatively higher accumulation in the abdominal region, potentially hinders the diagnosis of primary abdominal lesions [21]. In contrast, the reduced hepatobiliary excretion and comparatively lower abdominal uptake of [18F]AlF-FAP-NUR may facilitate the diagnosis of primary or metastatic lesions in the abdomen.

Our study has potential limitations. Firstly, our pilot study of [18F]AlF-FAP-NUR demonstrated promising imaging properties for cancer-associated fibroblasts but was limited by the number of patients available. However, a current investigation is underway to directly compare the [18F]F-labeled FAP peptide with [18F]FDG in a larger cohort of patients with various tumors. Secondly, directly substituting DOTA with NOTA without additional modifications may potentially result in suboptimal pharmacokinetic outcomes. In this study, we introduced a urea-modified tail at the N-terminus of the cyclic peptide, resulting in favorable pharmacokinetic profiles characterized by rapid renal clearance. However, gallbladder uptake remained high in human PET imaging. Despite this, fine-tuning the molecular structure or employing cutting-edge strategies such as CTR could potentially further improve the pharmacokinetics and distribution pattern of this tracer [18].

Conclusions

In conclusion, we developed a novel [18F]F-labeled FAP-targeting peptide using automated preparation for PET imaging of cancer-associated fibroblasts. [18F]AlF-FAP-NUR exhibited enhanced tumor-specific accumulation and high TBRs in FAP-positive mice, as well as demonstrated promising clinical PET/CT imaging results in cancer patients. These results highlight the potential of the tracer as a viable candidate for further clinical investigation. Further investigations are warranted to comprehensively elucidate the feasibility and efficacy of this tracer in a larger clinical cohort.

Data availability

The datasets used during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

Dr. Kongzhen Hu is acknowledged for providing the 293T-FAP and A549-FAP cells, and Dr. Zhihao Zha is acknowledged for providing the HT1080-FAP cells.

Funding

This research was supported in part by the National Natural Science Foundation of China (No. 82001879), the Applied and Basic Research Foundation of Guangdong Province (No. 2020A1515110159), the Science and Technology Project of Guangzhou City (No. 202102010354, No. 202201020558, No. 2024A03J1080), and the Zhongnanshan Medical Foundation of Guangdong Province (ZNSA-2020003).

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Author notes

  1. Shaoyu Liu and Ziqi Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of Nuclear Medicine, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510120, China

    Shaoyu Liu, Ziqi Zhang, Jiawei Zhong, Huizhen Zhong, Yimin Fu & Xinlu Wang

  2. Nuclear Medicine Department, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510655, China

    Lifang Liu & Xiaoting Ye

  3. Biomedical Innovation Center, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510655, China

    Lifang Liu & Xiaoting Ye

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Contributions

Conceptualization: SL, XW; Methodology: SL, ZZ, JZ; Validation: ZZ, JZ, XY, LL; Patients administration: HZ, YF; Data gathering and review: ZZ, SL; Formal analysis and investigation: SL, XW, ZZ; Writing - original draft preparation: SL, ZZ; Writing - review and editing: SL, ZZ, XW. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Xinlu Wang.

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This study was performed in line with the principles of the Declaration of Helsinki. All protocols of animal experiments in this study were approved by the Guangzhou Medical University Institutional Animal Care and Use Committee (No. 20230330), in compliance with the ARRIVE guidelines 2.0. The clinical translational study was approved by the institutional review board of the First Affiliated Hospital of Guangzhou Medical University (ES-2023-083-01), and written informed consent was obtained from the patient before the study. A statement to confirm that all experimental protocols were approved by First Affiliated Hospital of Guangzhou Medical University.

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Liu, S., Zhang, Z., Zhong, J. et al. Preclinical evaluation and first-in-human study of [18F]AlF-FAP-NUR for PET imaging cancer-associated fibroblasts. EJNMMI Res 14, 87 (2024). https://doi.org/10.1186/s13550-024-01139-w

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