Chemistry and radiochemistry
68Ga3+ was used under previously reported radiolabelling conditions to radiolabel FAPI-04 and generate FAP-targeting peptides with the chelator DOTA, with high radiochemical purity (> 95%), as validated by analytic radio-HPLC. The molar radioactivity of all tracers was estimated to be ~185.09 ± 13.5 GBq/μmol. The octanol/water partition coefficient (logP) was calculated as -2.92 (FAPI-04), -2.64 (FAPtp) and -2.88 (Alb-FAPtp-01), and the negative log p values indicate the high hydrophilicity of all tracers. Higher hydrophilicity is desirable for PET tracer design to reduce radioexposure, since the radioligands will quickly wash out through the urine. The proposed FAP tracers all demonstrated low liver uptake, which might be due to the PEGylating linker and highly hydrophilic peptide residues. The purity of all radiolabelled tracers was sufficient; thus, the tracers were passed through a 0.22 µm syringe filter for the following in vitro cell binding affinity assay or in vivo animal imaging studies without further purification.
To elucidate and quantify the kinetic binding ability of FAP with the proposed peptide tracers, a fast, real-time, high-throughput and label-free analysis method based on BLI was performed (Fig. 2). In these experiments, the association profile of FAP-04 was determined as an inhibitor, and the binding signals gradually increased over time and had a minimal dissociation drop pattern. The Kd value was determined as 17.4±3 x 10-6 M with BLI. However, the Alb-FAPtp-01 peptide tracers demonstrated the typical quick up-and-down profile pattern of small molecules with fast association and dissociation rates, and the Kd value was calculated as 18.5±2.4 x10-6 M. Due to the potential cleavage site (-Gly-Pro-) of FAP in the FAPtp peptides, the association rate of FAP is quite different than those the other two tracers. The association rate quickly increased, similar to Alb-FAPtp-01, at the early time points, but instead of reaching a plateau, the signals gradually increased over time but were not as high as those of FAP-04. This complex association pattern might be due to the peptide being cleaved by the FAP enzyme and cannot be simply determined by a 1:1 or 1:2 stoichiometry. These results were also confirmed with proteomic analysis experiments, in which the FAPtp peptide was found to be cleaved into two fragments when incubated with FAP enzymes, but the other two tracers were found to be intact under the same processing conditions (Supplementary Fig. S4). When DPP4 was used in this binding assay, only FAPI-04 demonstrated low binding and a quick dissociation profile, with a Kd value of 2.55 x 10-4 M (R2=0.96) (Fig. 2b). FAPtp and Alb-FAPtp-01 both demonstrated nonspecific binding throughout the experimental period. All these data indicated the specificity of the developed peptide tracers.
In vitro cell uptake and competition assays
The upregulated FAP expression in U87MG cells was confirmed by western blotting (Fig. 3 (a)) Then, the cellular uptake of [68Ga]Ga-FAPI-04, [68Ga]Ga-FAPtp and [68Ga]Ga-Alb-FAPtp-01 into U87MG cells was examined. In the cellular uptake experiment, U87MG cells were seeded into 24-well plates at a density of 0.5 million cells per well and incubated overnight. The cellular uptake was normalized to total added radioactivity. All tracers demonstrated comparable and specific cell uptake within 30 min of incubation and plateaued afterward, and [68Ga]Ga-Alb-FAPtp-01 showed slightly higher cellular uptake (> 2%) at 120 min but did not reach statistical significance (Fig. 3b). Note that the cellular uptake protocol used in this study does not distinguish between cell surface-bound and internalized radioactivity. In the competition study, the half maximal inhibitory concentrations [IC50] of [68Ga]Ga-FAPI-04, [68Ga]Ga-FAPtp and [68Ga]Ga-Alb-FAPtp-01 were 2.489 nM, 1.197 nM and 25.76 nM, respectively (Fig. 3c). The inhibition value of [68Ga]Ga-FAPI-04 was similar to that reported in previous papers ; however, a 10-fold higher concentration of FAPI-04 was needed to block the cellular uptake of [68Ga]Ga-Alb-FAPtp-01, indicating that Alb-FAPtp-01 may have a stronger ability to bind to U87MG cells than FAPI-04.
Dynamic PET imaging analysis of [68Ga]Ga-FAPI-04 and [68Ga]Ga-DOTA-FAPtp
Armed with promising data from in vitro assays, we were able to proceed with radiolabelling and evaluation of the FAP tracers in animal models with PET. [68Ga]Ga-FAPI-04 is believed to be a highly selective and potent FAP ligand (IC50 = 6.5 nM) and has emerged as a lead PET imaging agent [15-16] that might compensate for the functional role that has been dominated by 18F-FDG in recent decades. [68Ga]Ga-FAPI-04 has been proven to be highly correlated with FAP expression status clinically and was used as a reference tracer in this study. To validate the efficacy of the proposed [68Ga]Ga-DOTA-FAPtp tracer, 1-hour dynamic PET imaging of [68Ga]Ga-DOTA-FAPtp and potent FAP-targeting [68Ga]Ga-FAPI-04 tracers was acquired and compared side by side (see Supplementary Movies S1 & S2). After intravenous injection of both tracers, very good overall tumour radioactivity uptake and rapid whole-body washout patterns were observed (Fig. 4a). The time-activity curve of U87MG tumours displayed rapid peak uptake of both tracers, with approximately 1 standardized uptake value (SUV) by 10 min, but [68Ga]Ga-DOTA-FAPtp showed a delayed signal decline during the 20-40 min period, with a tumour uptake value of 0.5 SUV compared with 0.42 SUV for [68Ga]Ga-FAPI-04 (Fig. 4b). In addition to the higher tumour uptake observed for [68Ga]Ga-FAPtp, there was a much faster washout rate in the two major excretion organs, kidneys and liver, especially in the liver, and the clearance rate of [68Ga]Ga-FAPtp was 3-fold faster than that of [68Ga]Ga-FAPI-04, which may be due to the hydrophilic PEGylation linker of [68Ga]Ga-FAPtp that accelerated the tracer excretion time (Fig. 5). In addition, the higher heart accumulation signals indicated the longer plasma half-life of [68Ga]Ga-FAPtp. For brain and muscle tissues, the potential adverse side effects were minimal, as evidenced by indistinguishably low tracer uptake (SUV= 0.03 and 0.042, respectively) at late time points. These preclinical imaging results proved that the proposed FAP-targeting peptide tracer may be an effective positron emission tomography radioligand for quantifying FAP expression levels in U87MG tumours. The continuous washout of radioactivity occurred mostly through the renal pathway. Collectively, [68Ga]Ga-FAPtp peptide tracers bound reversibly to FAP and demonstrated comparable or even higher tumour targeting capability and more favourable biodistribution profiles than [68Ga]Ga-FAPI-04 and thus warrant further improvements in follow-up tracer development studies.
In vitro albumin binding affinity and in vivo pharmacokinetics of [68Ga]Ga-Alb-FAPtp-01
The major focus of this work was to modify and optimize current FAPI derivative-based tracers, such as FAPI-04, FAPI-21, and FAPI-46, to prolong tumour retention and improve the tumour-to-normal ratio based on the structural framework of the peptides, eventually making them potential theranostic agents. Therefore, we designed and synthesized prototypic [68Ga]Ga-FAPtp-targeted PET imaging agents with critical features of the peptide substrate and experimentally substantiated these agents. However, the prototype [68Ga]Ga-FAPtp tracer exhibited a rapid washout rate; thus, an intriguing in situ albumin binding strategy was incorporated into the tracer design with 4-p-chlorophenyl butyric acid. The concept of optimizing the tracer pharmacokinetic profile and prolonging tracer stability in the plasma has been proven to be effective for several published PSMA-relevant tracers. With adoption of the albumin-binding agent 4-p-chlorophenyl butyric acid, [68Ga]Ga-Alb-FAPtp-01 was first validated via in vitro FAP binding (Fig. 2), tumour cellular uptake (Fig. 3-b), albumin binding assays and in vivo half-life experiments (Fig. 6). The HSA binding affinity can reach a Kd value of 1.9 μM, and the half-life of the [68Ga]Ga-Alb-FAPtp-01 tracer in living animals was calculated to be approximately 120 mins, which reflected a significantly prolonged circulation time for such small peptide constructs that usually have circulation times of less than 10 min. Overall, these results demonstrated the high selectivity and albumin binding ability of the tracer, which may substantially enhance the opportunity for higher tumour uptake due to the long retention time in the plasma.
In vivo PET imaging evaluation of [68Ga]Ga-Alb-FAPtp-01
Therefore, intravenous administration of the optimized albumin binding [68Ga]Ga-Alb-FAPtp-01 tracer for PET/CT imaging was conducted to validate the FAP-targeting specificity. The imaging results showed profound rapid tumour uptake followed by moderately slow washout compared with the non-albumin-binding [68Ga]Ga-FAPI-04 and [68Ga]Ga-FAPtp peptide tracers (Fig. 7). The peak tumour uptake reached an SUV of 1.775 and subsequently slightly declined to 1.425 SUV at 180 min after injection. The percentage of radioactivity in the heart represented by the circulating tracers decreased to approximately 63.154±1.779% within the initial 30 min scan interval and thereafter slowly decreased to ~12.334±3.052% at the end of the scan (180 min), indicating that 4-p-chlorophenyl butyric acid played a role in optimizing the pharmacokinetics of the tracers. The tumour margins were visualized at early time points but may be confused with nonspecific perfusion imaging; thus, the persistent tracer signals detected at 2 or 3 hours post-injection in [68Ga]Ga-Alb-FAPtp-01 PET imaging reflect the specificity of tumour lesion detection. Furthermore, due to the low normal uptake background, the tumour contrast ratio of tumour/muscle increased significantly from 5.904±0.824 (1 hour) to 6.704±0.903 (2 hour) and 9.459±1.330 (3 hour), which indicates precise whole-tumour localization for preoperative planning and delineates the tumour margins from coregistered PET/CT imaging data with high sensitivity and high spatial resolution.
In addition, both the [68Ga]Ga-FAPI-04 and [68Ga]Ga-FAPtp tracers were quickly washed out through the kidney within 60 min. The higher blood circulation half-life of [68Ga]Ga-Alb-FAPtp-01 was attributable to the albumin-binding ability, which maintained a greater availability of radioligands in the plasma. Although there was somewhat higher liver uptake, the tumour uptake of the [68Ga]Ga-Alb-FAPtp-01 peptide tracer was significantly improved due to the prolonged and increased availability of stable radioligands in the plasma. These results validated the FAP-targeting specificity in animals injected with [68Ga]Ga-Alb-FAPtp-01 tracers.
Longitudinal monitoring of tumour growth with FAPI-targeted PET imaging
An elevated FAP expression level in tumour neoplasia was correlated with high tumourigenic and metastatic potential and a high rate of mortality in clinical patients, justifying its use as a potential prognostic marker. To further confirm the application of imaging with tracers for staging, longitudinal PET scans were conducted. The stable [68Ga]Ga-Alb-FAPtp-01 PET tracer demonstrated prominent longitudinal tumour uptake in subcutaneous U87MG xenografts with sequential imaging acquisition time points at different tumour sizes/stages (tumour size from 200-870 mm3). The tracer uptake in malignancies was 0.540+/-0.096, 1.198+/-0.155 and 1.829+/-0.100 (SUVmean) at the small, medium and large stages (late stage), respectively. The increased tumour uptake observed in PET/CT imaging was consistent with the immunostaining profile of FAP, and tumour growth was accompanied by upregulation of FAP expression levels. Overall, the developed albumin-binding FAP-targeting [68Ga]Ga-Alb-FAPtp-01 tracer was able to differentiate the malignant neoplasia of U87MG tumours, and this feature may serve as a prognostic marker to identify the specific tmour phenotype for anti-FAP therapies. These pilot studies demonstrated that [68Ga]Ga-Alb-FAPtp-01 tracers may be superior to FAPI-04 PET for phenotypic tumour imaging for patient distraction diagnosis. Furthermore, it is worth noting that albumin-binding FAPtp-01 tracers are more suitable for treatment response monitoring, with potential applications for anti-FAP interventions or combination therapy with conventional treatments, such as radiotherapy and chemotherapy drugs. These follow-up studies are currently being performed.
The in vivo three-dimensional (3D) PET imaging is presented as a movie (see supplementary S3), and the PET imaging was strongly correlated with ex vivo autoradiography images, suggesting that FAP can be accurately validated by the proposed [68Ga]Ga-Alb-FAPtp-01 PET tracer (Fig. 9). We next explored the relationship between radioactivity signal images and the ex vivo histopathologic analysis of FAP and vascular maturity (CD31-positive blood vessels) on a per-tumour basis in tumour xenograft models (Fig. 9b). U87MG xenograft tumours were collected and stained with antibodies against FAP and CD31 to evaluate FAP and vascular density by histochemistry. The PET imaging and ex vivo autoradiography images were compared spatially with the immunostaining data. The results demonstrated that the vascular density and resulting tumour perfusion were not directly correlated with [68Ga]Ga-Alb-FAPtp-01 tracer uptake, as shown in Fig. 9. After investigating the histopathologic data in greater depth, it was obvious that the tumour regions with higher FAP expression levels but relatively low vascular density (weaker CD31 immunostaining) have significantly high tracer uptake in both in vivo 3D volume-rendered PET imaging and ex vivo autoradiography. The spatial colocalization and cooccurrence of the histopathologic staining with tracer accumulation confirmed the accurate registration of FAP expression status. However, vascular function and perfusion status were not significantly related to tracer accumulation but to FAP-binding specificity. Overall, in addition to FDG, the albumin tracer [68Ga]Ga-Alb-FAPtp-01 might provide complementary information for the noninvasive assessment of tumour status and thus support better patient stratification and subsequent monitoring of therapeutic response.