Translational assessment of a DATA-functionalized FAP inhibitor with facile ⁶⁸Ga-labeling at room temperature

Most tumors develop as solid tissue masses with a distinct structure, encompassed by two strongly associated compartments: neoplastic cells and stroma. Stroma is induced by the neoplastic cells which are further dispersed in it. For the tumors to grow above a minimum size of 1 to 2 mm, the creation of stroma is necessary since it mainly includes connective tissue, the basement membrane, fibroblasts, extracellular matrix, immune cells, and blood vessels. Even though stromal components hold certain tumor-suppressing abilities, stroma appears to change during the malignancy. Eventually the interactions between the cancer and stromal cells promote growth, invasion, and metastasis. The increased number of fibroblasts contained in stroma are pathologically activated within the course of the disease, forming the so-called cancer associated fibroblasts (CAFs) [1]. CAF activation may be influenced by several stimuli, both cellular and environmental, leading to a variety of CAF phenotypes [2], that are identified by several biomarkers expressed on their surface [3]. CAFs appear to have an abundant expression of fibroblast activation protein (FAP), also known as prolyl endopeptidase FAP or seprase, associated with fibrosis, tissue repair, inflammation, and extracellular matrix (ECM) degradation. FAP is also involved in tumor growth, invasion, metastasis and immunosuppression [4].

FAP is overexpressed in malignant tissue on activated fibroblasts on a variety of malignant tumors such as breast, colorectal, pancreatic, melanoma, myeloma, gastric, brain, and ovarian carcinomas [5‐11] while it shows less abundance in normal healthy tissue. Hence, FAP radioligands are considered important tools for in vivo tumor imaging and/or therapy [12].

With the view to develop pan-cancer theranostic radiotracers, several research groups have shifted their scientific focus on imaging and/or treating various tumors by targeting not directly the cancer cells but FAP⁺ CAFs [1]. Many efforts have been made in the last years, and several FAP-targeting radiotracers, derived from antibodies, FAP-inhibitors (FAPi) and peptides, have shown great promise in preclinical [13‐26] and clinical settings [13‐15, 20, 21, 23, 27‐35]. The FAP inhibitor UAMC1110, due to its high potency and selectivity towards FAP, appears to be a highly suitable candidate for the development of FAP-based radiopharmaceuticals [36]. For radiolabeling with radiometals, UAMC1110 is functionalized with a matching chelator. Although, in principle, many different chelators are available for gallium-68, the chelator DATA^5m is particularly attractive since it can be conveniently labeled at room temperature (RT) [37]. Coupling of DATA^5m to UAMC1110 via a squaric acid based spacer has resulted in the lead candidate DATA^5m.SA.FAPi (Fig. 1) [18, 19, 38].

This study aimed at determining a set of preclinical characteristics of [⁶⁸Ga]Ga-DATA^5m.SA.FAPi, which appeared to be relevant in the course of the ongoing clinical applications [39‐41] of this tracer:

We evaluated [⁶⁸Ga]Ga-DATA^5m.SA.FAPi in vitro including stability studies, metabolite analysis, protein binding, and internalization studies. [⁶⁸Ga]Ga-DATA^5m.SA.FAPi was further evaluated in vivo and ex vivo in glioblastoma and prostate xenografts by biodistribution and PET/CT imaging studies. Further on, the PET/CT data from six patients suffering from metastasized castration resistant prostate cancer (mCRPC) were analyzed to compare small-animal and patient uptake profiles. In preparation of therapeutical applications, biodistribution, and biokinetics in the organs potentially at risk when using [⁶⁸Ga]Ga-DATA^5m.SA.FAPi were determined including dosimetry.

The supplier information for all reagents and details of instruments used are provided in the supplemental data.

[⁶⁸Ga]Ga-DATA^5m.SA.FAPi, previously reported by Moon et al., provided affinity and selectivity data towards FAP [18, 19]. Herewith, our goal was to evaluate the performance of [⁶⁸Ga]Ga-DATA^5m.SA.FAPi by investigating, preclinically and clinically, its ability to detect FAP-positive tumors of different origin.

The coupling of DATA^5m via the squaric acid-based spacer to UAMC1110 and the radiolabeling with gallium-68 did not influence UAMC1110 high FAP binding affinity as well as its in vivo stability. In our saturation binding assay, ^68/natGa-DATA^5m.SA.FAPi showed extremely high affinity towards FAP on CAFs, with a K_d value in the sub-nanomolar range. Additionally, more than 95% of the radioactivity in human serum corresponds to intact radiotracer indicating that the radiotracer is not subjected to in vivo metabolic degradation. The enhanced affinity towards FAP may have also resulted in the fast internalization rate.

[⁶⁸Ga]Ga-DATA^5m.SA.FAPi, most probably due to its balanced lipophilicity, delivers biodistribution and PET images which advocate specific uptake by both experimental tumor. Elimination took place mainly via the urinary tract with a lower residence in the kidneys and higher in the urinary bladder wall both in mice and humans. UAMC1110 small size might be responsible for the low kidney uptake, which seems to be a superiority of the UAMC1110-based radiotracers compared to the peptidomimetic-based FAP targeting counterparts where it was shown that kidney was the organ with the highest non-target uptake at early time points [24]. The gradually increasing uptake in the gallbladder, as indicated by PET images, may also suggest a second excretion pathway. Another point, which is in accordance with the findings for other radiolabeled FAP inhibitors concerns the non-specific accumulation of [⁶⁸Ga]Ga-DATA^5m.SA.FAPi in non-target organs including muscles, lung, pancreas and joints [13‐15, 17, 20, 21]. This context suggests that fibroblasts involved in the development of those organs may also be detected by FAP targeting. Additionally, the relatively high blood pool uptake by the radiolabeled FAP- inhibitors in general is one of the challenges. Studies have shown the presence of a soluble FAP form in both bovine [42] and human plasma [43]. Up to date, it is not clear which is the function of this soluble form; if, for example, there is any interaction with the full-length membrane-bound FAP form or whether the presence of the soluble FAP form is the result of shedding from the membrane surface or the biosynthesis of alternative splicing. The understanding of how FAP enters the circulation but also how and to which extent, FAP targeting might be influenced when using nuclear probes, is of paramount importance to strengthen the development of FAP-based radiotracers and improve the quality of the current nuclear imaging applications.

The PC3 and U87MG subcutaneous xenografts were used because of their modest tumor growth and the abundant FAP expression in the stroma of human prostate carcinomas [27, 28] and glioblastomas [22, 44]. However, in cancer cell-derived xenografts, tumor growth requires the development of tumor microenvironment and the recruitment of murine fibroblasts. Due to the similar functional homology and the 89% shared sequence identity between murine and human-FAP, studies have shown that murine-FAP also promotes tumor growth in cell derived xenografts [45]. Our in vitro data showed low FAP protein expression for U87MG and even lower for PC3 cells compared to CAF. The radioligand binding assays confirmed high FAP cell-surface expression for CAF, very low for U87MG and negligible for PC3. However, under in vivo conditions, where neither extended vascularization nor blood pool activity in the tumors was observed, the radiotracer is specifically taken up by both experimental tumors. Further studies are required to verify if FAP cell-surface expression is limited to the murine stroma, or if the initially FAP-surface low expressing (U87MG) or negative tumor cells (PC3) turned out to be FAP-positive when exposed to murine CAFs. The murine origin of tumor stroma in xenotransplanted mice is certainly a limitation since the tumor environment and molecular signature of the corresponding human tumor are not retained. Nevertheless, the successful FAP targeting by [⁶⁸Ga]Ga-DATA^5m.SA.FAPi provides a strong evidence of the otherwise successful strategy of tumor stroma targeting.

Patient-derived xenografts (PDXs) might represent a more suitable model for FAP targeting in preclinical settings, however, special attention should be paid, since only the PDXs established by direct implantation of fresh surgical tissue fragments into mice mimic the human conditions. After a couple of passages murine fibroblasts are recruited and tumor stroma is eventually dominated by them.

The encouraging in vitro and in vivo preclinical data of [⁶⁸Ga]Ga-DATA^5m.SA.FAPi was the driving force to immediately plan and execute a study aiming on acquiring initial clinical experience on human prostate cancer patients and verifying if the preclinical data are translated to humans. These data were also collected as part of the preparation for potential FAPi-based therapy. [⁶⁸Ga]Ga-DATA^5m.SA.FAPi is mainly eliminated via the urinary tract. Considerable uptake in organs presenting FAP expression and tumor lesions was observed, which makes the images easy to interpret. The optimum time for PET/CT appears to be 40–50 min after the administration of the tracer. Interestingly, the absolute tumor uptake values in animals (% activity/gram; Table 2, Fig. 5) are slightly lower in patients (SUV_max; Fig. 7). More importantly, the tumor-to-organ and tumor-to-background ratios, respectively, are much higher in patient studies compared to the preclinical data. These non-mirroring findings may be due to the mixture of both species, human- and murine-FAP on the experimental tumors. The fact that the small animal PET modalities have not exactly the same technical specifications with the clinical scanners might be a second reason for the non-direct translation of preclinical imaging findings to clinical systems.

The clinical biodistribution and kinetics data published for [⁶⁸Ga]Ga-DATA^5m.SA.FAPi [39‐41]are comparable to other FAPi tracers [29, 32, 46]. As with these, the high tumor-to-background ratio achieved after only 10 min enables early imaging with [⁶⁸Ga]Ga-DATA^5m.SA.FAPi, with the contrast ratio still improving over time. This opens a wide time window for imaging, which can thus be adapted to clinical needs. The average effective dose for the administration of 200 MBq of [⁶⁸Ga]Ga-DATA^5m.SA.FAPi was 2.7 mSv, which is slightly higher compared to the reported values for other FAPi tracers (e.g. 1.56 mSv and 2.2 mSv for [⁶⁸Ga]Ga-FAPI-46 [46] and [⁶⁸Ga]Ga-DOTA.SA.FAPi [32], respectively), but without being associated with a higher risk in use. In principle, the results of incorporation dosimetry are comparable to those of other ⁶⁸Ga-labelled tracers, e.g. tracers targeting prostate-specific membrane antigens or somatostatin receptors [47‐49].

In conclusion, the preclinical and clinical data presented in this work, in particular also the data related to calculation of the incorporation dosimetry for [⁶⁸Ga]Ga-DATA^5m.SA.FAPi, will support further clinical application of this DATA^5m.SA-functionalized FAP inhibitor with superior radiolabeling properties. One main advantage of DATA^5m.SA.FAPi compared to other reported FAPi-based precursors is due to its functionalization with the chelator DATA^5m which allows the preparation of the ⁶⁸Ga-labelled radiotracer in an “advanced” kit-type protocol since fast and efficient radiolabeling can be achieved at ambient temperature [37].