Fibroblast activation protein (FAP) is a single-pass type II transmembrane glycoprotein (1), with a large extracellular domain composed of an α/β-hydrolase and an 8-bladed β-propeller (2). As a member of the S9 prolyl oligopeptidase family, FAP has post-proline exopeptidase activity similar to dipeptidyl peptidase 4 (DPP4), and an additional endopeptidase activity like prolyl endopeptidase (PREP) (3,4).
FAP is expressed during embryonic development, with highly restricted expression in healthy adult tissues (5,6). However, FAP upregulation can occur in diseases associated with activated stroma including wound healing, rheumatoid arthritis, cirrhosis, pulmonary fibrosis, and solid cancers (7–10). In most epithelial cancers, FAP is selectively expressed on the cell surface of cancer-associated fibroblasts (CAFs) present in the tumor microenvironment (11). These CAFs play important roles in tumor growth and metastasis through their effects on angiogenesis, the extracellular matrix, and the immune system (12). In addition, in several tumors of mesenchymal origin, notably sarcoma and mesothelioma, FAP expression has also been observed on the neoplastic cells themselves (11) and is similarly associated with promoting tumor progression and metastasis (6). Given its restricted expression profile and functions, FAP is a promising target for the selective delivery of anticancer therapies to tumors of a broad range of cancer indications (13–16).
Many FAP-targeting agents have been developed including small molecule inhibitors and immunoglobulins. Of the antibody-based therapies, sibrotuzumab has been evaluated in a clinical trial and demonstrated no objective clinical responses, while the antibody drug conjugates FAP5-DM1 and OMTX705 have yet to be investigated in patients (16,17). In addition, the radioimmunoconjugate ESC11 designed for diagnostic and therapeutic applications has shown efficacy in a preclinical melanoma xenograft model (15). However, antibodies are well known to have an extended blood circulating half-life which may elicit greater normal tissue toxicity, particularly in the bone marrow when conjugated to a radionuclide. In contrast, small molecular weight radioconjugates have the advantage of rapid tumor delivery coupled to fast systemic clearance to potentially circumvent this toxicity.
One approach to targeting FAP is through radiolabeled theranostic agents, integrating both noninvasive disease diagnosis and treatment. Validated targeted radiopharmaceuticals include LUTATHERA® (177Lu-dotatate; Advanced Accelerator Applications), a cyclic peptide targeting somatostatin subtype 2 receptor (SSTR2) armed with the β-emitting radionuclide lutetium-177 (177Lu), which has been approved for treatment of SSTR2-positive patients with gastroenteropancreatic neuroendocrine tumors (GEP-NET) (18); and 177Lu-PSMA-617, a small molecule targeting prostate-specific membrane antigen (PSMA) linked to 177Lu, which recently met both primary survival endpoints in the phase III VISION trial for PSMA-positive metastatic castration-resistant prostate cancer (mCRPC) (19).
FAP is an emergent target in the radiopharmaceutical field and has been referred to as “the next billion dollar nuclear theranostics target” (20). This description originates from the development of the FAP inhibitor (FAPI) series of small molecule quinoline-based radiotracers (21) that have shown high specificity across various primary and metastatic tumors (22,23). The diagnostic utility of 68Ga-FAPI–based positron emission tomography (PET) has been established in various cancer types, demonstrating substantial accumulation in cancer of unknown primary (CUP), sarcoma, esophageal cancer, breast cancer, and cholangiocarcinoma (22). In addition, 68Ga-FAPI-04 appears to outperform 18F-fluorodeoxyglucose (18F-FDG) in discriminating the primary and distant metastatic lesions in certain indications (24–26).
Further structural modifications of the FAPI series of compounds are currently ongoing to improve pharmacology and allow the attachment of alternative radionuclides (27,28). In parallel, multiple academic and commercial entities have recently described highly related FAP-targeting molecules with similar results (29,30), including the lead therapeutic compound FAPI-46 with a methylamino alteration in the linker region. However, the FAPI radiotracers are established around UAMC1110 (31), a small molecule inhibitor of FAP, and exhibit fast clearance from tumors, limiting their potential therapeutic effectiveness.
To address the low tumor retention of the FAPI series, we developed the FAP-2286 compound, based on a novel class of FAP-targeting modalities, that utilizes cyclic peptides as binding motifs. Cyclic peptides are known to have potentially improved biological properties over linear counterparts, including greater binding affinity and selectivity due to their conformational rigidity and increased plasma stability. FAP-2286 is composed of such a cyclic peptide, which is linked to a tetraazacyclododecane tetraacetic acid (DOTA) allowing chelation of radionuclides for imaging or therapeutic applications. For imaging, the radionuclides gallium-68 (68Ga) or indium-111 (111In) were chelated to FAP-2286 for use as a PET or single-photon emission computed tomography (SPECT) imaging agent, respectively. For therapeutic use, 177Lu was chelated to FAP-2286. Herein, the in vitro and in vivo characterization of FAP-2286 and its metal complexes are reported and compared to FAPI-46.
Material and methods
Detailed information on materials and methods including peptide synthesis are described in the Supplementary Materials
Formalin-fixed paraffin-embedded (FFPE) tissue microarrays (US Biomax) and whole sections (Indivumed, Tissue Solutions, and Discovery Life Sciences) were stained with FAP antibody (SP325, Abcam; 1:50) according to the manufacturer recommendation. Staining was performed on the Bond Rx Autostainer (Leica Biosystems) and detection by the Bond Polymer Refine Detection (Leica Biosystems) according to manufacturer’s protocol. Whole slide scanning was performed on the Aperio AT2 (Leica Biosystems), and analyses were conducted using Aperio ImageScope (Leica Biosystems), Visiopharm Integrator System (Visiopharm), or HALO (Indica Labs) image analysis platform.
In vitro FAP autoradiography was performed as previously described with minor modifications (32). The 20-µm-thick frozen tissue sections were incubated with 111In-labeled FAP-2286 (15 MBq/nmol). Subsequently, an x-ray film (Biomax MR films; Carestream Health) was placed onto positioned slides and exposed for 2 days. Relative optical film density was determined using MCID™ software (InterFocus) and correlated with known amounts of radioactivity via a separately recorded calibration curve.
In vitro assays
Surface plasmon resonance assay (SPR)
The binding kinetics of FAP-2286 to antibody-immobilized human FAP (Sino Biological) or mouse FAP (R&D Systems) was measured by SPR and calculated using single-cycle kinetic measurements.
Cell-based binding assay
FAP-expressing WI-38 fibroblasts (ECACC) were co-incubated for 1 h at 4 °C with Cy5-labeled FAP-binding competitor peptide and various concentrations of FAP-2286. Median fluorescence intensity was measured by flow cytometry on Attune NxT (Thermo Fisher Scientific), and 4PL curve fitting and half-maximal inhibitory concentration (IC50) calculations were performed using the ActivityBase software (IDBS).
Protease activity assay
Recombinant human and mouse FAP (R&D Systems) were incubated with various concentrations of FAP-2286 before a fluorophore-labeled specific substrate (Eurogentec; (33)) was added to the well, and fluorescence output was measured for 5 min at 37 °C on SpectraMax M5 (Molecular Devices).
Plasma stability assay
FAP-2286 was incubated in human or mouse plasma at 37 °C for 24 h. Compound concentration was measured using HPLC–MS/MS (Agilent Technologies) and percent concentration remaining was calculated.
Murine tumor models
HEK-FAP cell line was generated using the vector pTAR to insert FAP complementary DNA by recombinase-mediated cassette exchange into the pre-tagged HEK293 cells (InSCREENeX). For in vivo HEK-FAP studies, female nude or SCID beige mice (Charles River Laboratories) were subcutaneously implanted with 5 × 106 HEK-FAP cells in 150 µL serum-free media:Matrigel (1:1) (Corning). For the patient-derived xenograft (PDX) efficacy study, female NMRI nu/nu mice (Janvier Labs) were subcutaneously implanted with Sarc4809 fragments (Experimental Pharmacology & Oncology Berlin-Buch). All animal experiments were carried out under licenses approved by the respective authorities and in compliance with guidelines on animal welfare.
In vivo biodistribution
Thirty megabecquerels (30 MBq/nmol) 111In-FAP-2286 (n = 9) or 111In-FAPI-04 (n = 6) was injected intravenously (IV) into HEK-FAP tumor-bearing mice and the distribution of the radiotracers was assessed using the NanoSPECT/computed tomography (CT) system (Mediso). For PET imaging, 10 MBq of 68Ga-FAP-2286 or 68Ga-FAPI-46 (10 MBq/nmol) was given IV into HEK-FAP tumor-bearing mice (n = 3) followed by PET/CT analysis using the nanoScan PET/CT system (Mediso). For SPECT imaging of 177Lu-labeled FAP-2286 or 177Lu-labeled FAPI-46, 30 MBq was administered IV (30 MBq/nmol; n = 3) and the distribution of the radiotracers was assessed using the nanoScan SPECT/CT system (Mediso).
A single dose of the indicated treatments was administered IV on day 0, with vehicle (saline), nonradioactive natLu-FAP-2286, 30 or 60 MBq 177Lu-FAP-2286 (30 MBq/nmol and 60 MBq/nmol, respectively), or 30 MBq 177Lu-FAPI-46 (30 MBq/nmol). Tumor length and width were measured by caliper, and tumor volume was calculated as 0.5 × (length × width2). Tumor growth and body weight were monitored 3 times weekly. Sarc4809 tumor growth was normalized to the mean tumor volume (MTV) at the time of injection for each group due to variability in tumor volume between the groups. Statistical analysis was performed using GraphPad Prism 8.4.
Cellular internalization assay
FAP-2286 was labeled with Alexa Fluor 488 (AF488) at the C-terminus of the peptide. For FAPI-46, a free carboxy moiety of the DOTA was used for labeling using AF488 Cadaverine (Thermo Fisher Scientific).
HEK-FAP cells were incubated with various concentrations of AF488-labeled FAP-2286 or FAPI-46 for 1 h at 37 °C. Median fluorescence intensity (MFI) was measured on Attune NxT (Thermo Fisher Scientific).
HEK-FAP cells were seeded on chamber slides (ibidi) and incubated with 5 nM of AF488-labeled FAP-2286 or FAPI-46 for 1 h at 37 °C. Cells were then washed and further incubated for various timepoints. Cell nuclei were stained with 1 µg/mL Hoechst 33342 dye. Lysosomes were stained using LysoTracker Deep Red (Thermo Fisher Scientific). Images were acquired by using the BZ X800E fluorescence microscope (Keyence) at 40 × magnification.
FAP is an attractive cell surface target for radiopharmaceutical development given its tumor-restricted expression profile. While typically low to undetectable in most normal adult tissues, FAP is highly expressed on the cell surface in multiple cancer subtypes (6). In this study, the abundant expression of FAP as determined previously by others through mRNA and protein analyses was confirmed in tumor tissues of diverse origin, including pancreatic ductal adenocarcinoma, sarcoma, CUP, mesothelioma, cholangiocarcinoma, and breast cancer. While largely consistent across studies, some minor differences between FAP protein expression derived from TMA versus the larger tissue section immunohis to chemistry results were observed. For example, bladder cancer sections demonstrated stronger staining with median H-score of 25.0 (38% FAP positive, n = 26) than the TMA cores with 2.5 median H-score (n = 40). This discrepancy may be due to the metastatic bladder specimens assessed as whole sections (n = 21), whereas TMA bladder cores were from primary lesions; also, FAP expression in bladder metastasis has been reported to increase in immunotherapy-resistant tumors (35).
For most tumor specimens examined, FAP expression was restricted to the CAFs in the stromal cell compartment of the tumor. In comparison, in a large subset of sarcomas and mesotheliomas and smaller subset of epithelial derived cancers including esophageal, FAP expression was observed in tumor cells in addition to the CAFs population. These results are consistent with studies reporting tumors of mesenchymal origin express FAP in cancer and stromal cell populations (11,36). Furthermore, FAP expression as analyzed by immunohistochemistry directly correlated with data obtained by autoradiography of matched tumor specimens using 111In-FAP-2286 as the detection reagent, suggesting that FAP-2286 binds specifically to FAP.
Low-molecular-weight targeting approaches for radiotherapy, including small organic molecules and peptides, have the advantage of rapid tumor delivery and systemic renal clearance, augmenting efficacy and reducing the potential for myelosuppression. Of these compounds, the quinoline-based FAPI series of radiotracers have demonstrated compelling imaging results in patients (21–23,25). However, because of their short tumor residence time, FAPI radiotracers in their current form are not optimal for therapeutic applications when utilizing radionuclides with a half-life > 24 h. Currently validated small molecule radiotherapeutics such as LUTHERA® (177
Lu-dotatate) and PSMA-617 employ 177
Lu as a β-emitting radionuclide, which has a half-life of 6.7 days, to deliver a persisting radiation dose for therapeutic applications. Using the HEK-FAP mouse tumor model, we confirmed the lower tumor retention of FAPI-04 with a rapid clearance rate of approximately 50% every 24 h (Supplementary Fig. S6
), consistent with fast washout kinetics reported previously (21,28,37).
To overcome the short retention time, second-generation molecules based on the same FAP-targeting moiety of UAMC1110 (31) employed in FAPI series of compounds have been developed including QCP02 (30), RPS-309 (38), and OncoFAP (29,39). While most are in preclinical testing, 2 radiotracers have been evaluated in patients as imaging agents: DOTA.SA.FAPi and FAPI‐46. With a squaramide linker for improving biological half-life, DOTA.SA.FAPi has demonstrated similar performance as 18F-FDG in diagnosing patients (40). Likewise, FAPI‐46, as a continuation on the FAPI series, showed improved tumor‐to‐organ ratios, resulting in enhanced image contrast (28). However, the increase in tumor retention time for FAPI-46 and DOTA.SA.FAPi was incremental, and further molecular modifications may be required to allow the optimal use of isotopes with long decay rates for therapeutic applications.
FAP-2286 was developed with the hypothesis that an improved tumor retention time could be achieved using a cyclic peptide to target FAP compared to small molecule–based radiotracers. FAP-2286 labeled with 177Lu displayed prolonged tumor retention (≥ 72 h) compared to FAPI-46 after injection in the HEK-FAP mouse model as measured by SPECT/CT image quantification, and yet maintained rapid system renal clearance as expected for small molecules. The longer tumor retention of FAP-2286 as compared to FAPI-46 resulted in 12- and 9-fold higher TIAC and absorbed dose delivered to the tumors, respectively, which ultimately lead to greater tumor inhibition.
Consistent with the SPECT imaging data, Alexa Fluor 488–derivatized FAP-2286 was retained in HEK-FAP cells and secluded in endosomes out to 72 h in vitro, whereas Alexa Fluor 488–derivatized FAPI-46 levels decreased over time starting at 8 h, despite both showing equivalent level of initial binding to HEK-FAP cells. A previous study reported similar results with a first-generation FAPI compound (FAPI-02) in HT1080-FAP cells (41). In binding and efflux experiments, 177Lu-FAPI-02 retained 34% of its initial radioactivity after 24 h of incubation, which was later demonstrated to be comparable to FAPI-04 (21).
The benefit of the long retention of FAP-2286 was observed not only in HEK-FAP but also in Sarc4809 PDX tumors after a single administration of 177Lu-FAP-2286. Compared to the vehicle control group, statistically significant antitumor activity evident by tumor shrinkage was observed from day 5 onward in the HEK-FAP mice of high- and low-dose treatment groups. Similarly, in the Sarc4809 PDX model of sarcoma, there was a statistically significant difference in the RTV between the high- and low-dose treatment versus control groups on day 19. The observed therapeutic effect in the treatment groups is attributed to the targeted delivery of the β-particle emitting radionuclide 177Lu to the tumor mass, since the nonradioactive natLu-labeled compound had no effects. Tumor suppression is therefore presumed to be caused by radiation-induced DNA damage and subsequent apoptotic cell death of the FAP-expressing cells (42,43). In this scenario, the tumor model Sarc4809 would be representative of cancers of mesenchymal origin, where FAP is present on tumor cells contributing to their histologies and tumorigenesis (11) and 177Lu-FAP-2286 would render cytotoxic radiation to tumor cells.
Targeting FAP is not limited to tumor cells but can also be applied to the CAFs in the stroma that support tumor development and maintenance (6). In cancers of epithelial origin, FAP expression is mainly restricted to the CAFs in the tumor microenvironment, which possesses diverse functions, such as stromatogenesis, reciprocal signaling interactions with cancer cells, and crosstalk with tumor-infiltrating leucocytes (6,12). Because FAP-expressing CAFs play an essential role in tumorigenesis, their depletion has been associated with tumor shrinkage (44–46).
In addition to CAF-directed ablation by the β-particle emitting radionuclide payload, radiation-induced bystander effects and crossfire targeting of tumor cells can occur in the vicinity of CAFs, where the radiopharmaceutical accumulates (47). Tumor cells in the bordering areas adjacent to FAP-expressing CAFs are affected by radiobiological damage as a result of signals originating from irradiated cells (48), while the crossfire effect occurs within the range of the β-particles by traversing radiation penetrating several cell layers (49). Consequently, the radiotherapeutic approach using β-particles may be advantageous in terms of a broader destruction of the tumor mass over other FAP-targeting agents that only directly affect FAP-expressing CAFs. Further work using mouse tumor models with FAP-positive CAFs infiltration is warranted, and model development studies are on-going to recapitulate CAFs recruitment and maintenance in mice. However, due to the lower affinity of FAP-2286 towards murine FAP the utilization of a surrogate molecule with higher binding to murine FAP would be needed to enable translating the results to humans.
Apart from kidney uptake, which was related to the excretion of the radiolabeled peptide and rapidly cleared, limited accumulation of radioactivity in other non-target tissues was observed during imaging. This resulted in the high HEK-FAP tumor-to-background contrast in the 177Lu-FAP-2286 SPECT/CT images without treatment-related acute toxicity or body weight loss in the efficacy studies. However, the studies described presently were not designed to assess the long-term hematologic and renal toxicities or the secondary malignancies that have been reported in patients with targeted radiotherapies (50).
In the first-in-human experience of 11 patients, FAP-2286 has established its utility as a theranostic agent labeled with 68Ga for diagnosis and with 177Lu for treatment. PET/CT scans of 68Ga-FAP-2286 confirmed significant uptake in neoplastic lesions and low presence in normal tissues of patients with either pancreatic, breast, ovarian, or colorectal carcinomas. As a therapeutic radiopharmaceutical, 177Lu-FAP-2286 showed similar biodistribution by SPECT/CT as the imaging agent 68Ga-FAP-2286, and likewise, it had significant tumor uptake and long retention that appeared to improve symptoms manifested by pain reduction in 3 patients with advanced disease (51).
Taken altogether, FAP-2286 demonstrates compelling characteristics of a targeting agent with potent and selective FAP binding that leads to high tumor accumulation and substantial therapeutic efficacy. The preclinical studies as well as the first-in-human experience support further development of 68Ga-FAP-2286 and 177Lu-FAP-2286, and evaluation in patients with advanced solid cancers in the ongoing phase 1/2 clinical study LuMIERE (NCT04939610) for assessment of their safety, pharmacokinetics, dosimetry, and efficacy.
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