In the recent years, the development of tumor targeting ligands resulted in the discovery of new generation diagnostic and therapeutic products with high uptake in cancer lesions and low accumulation in healthy organs. Lutathera® and 177Lu-PSMA-617 provide examples of radioligand therapeutics with proven clinical efficacy and limited systemic toxicity [13, 14].
Radioligand diagnostic and therapeutic products targeting Fibroblast Activation Protein (FAP) represent a promising class of compounds with pan-tumoral applicability and high selectivity for tumor lesions. FAP is an extracellular protease, highly expressed on the cell surface of cancer-associated fibroblasts (CAFs). Being among the major cell populations within the stroma of solid tumours, cancer-associated fibroblasts (CAFs) provide physical support for tumour cells and exert protumorigenic functions, modulating disease progression and resistance to therapies . In this context, targeting stromal structures may represent therapeutic advantages over approaches directed against cellular antigens like PSMA and somatostatin receptors .
FAP-targeting strategies have gained growing relevance in nuclear medicine for the development of radiotheranostics. Although anti-FAP antibodies have been known since the 1990s [17–20], the discovery of small organic FAP ligands (the so-called “FAPI” tracers) in the last few years represented a significant improvement in the field. Among FAP small organic ligands, OncoFAP is the one with the highest affinity described so far (Kd = 680 pM) . Various FAP-targeting agents coupled to different radionuclide chelators have been recently developed and characterized for their high affinity and selectivity towards FAP. These conjugates exhibit a rapid accumulation in cancer lesions and low uptake in healthy organs both in preclinical murine models and cancer patients. However, the translation of radiopharmaceutical agents from preclinical development to clinical applications requires the implementation of appropriate non-clinical procedures, including recommendations for standard and automated radiolabelling procedures.
In this work, we have described the development of robust and automated methodologies for the efficient radiolabelling of OncoFAP-derivatives (OncoFAP-DOTAGA, OncoFAP-NODAGA and OncoFAP-NOTA) with 68Ga, Al18F and 177Lu radioisotopes. Methodologies described here are novel, easy to implement, safe and robust. We based these procedures on the FASTLab2 automated module, a radiolabelling platform already broadly applied to produce other radiopharmaceuticals at the commercial scale [21–23].
With the aim to enable production of [68Ga]-OncoFAP-DOTAGA/NOGADA radiopharmaceuticals also in small radiopharmacy sites with limited financial resources, we have created and validated a radiolabelling single-vial cold kit. This approach, already proposed for other products (Illuccix™ and NETSPOT®; SOMAKIT TOC) [12, 24, 25], renders the production of 68Ga-based products as easy as the one of 99mTc-based radiopharmaceuticals. The “OncoFAP-kits”, which already contains all necessary items (buffers and ligands) to be combined with the radioisotopes, allow to efficiently afford [68Ga]Ga-OncoFAP-NODAGA and [68Ga]Ga-OncoFAP-DOTAGA for PET imaging applications.
OncoFAP-DOTAGA can be labelled both with 68Ga and 177Lu, and thus can be used as a precursor of a “theranostic pair”. This approach has been already successfully implemented for the diagnosis and therapy of Non-Endocrine Tumours (Lutathera®/NETSPOT®)  and prostate cancers (68Ga-PSMA-11/177Lu-PSMA-617) . [177Lu]Lu-OncoFAP-DOTAGA was obtained with specific activities of about 2.5 mCi/µg, which almost doubles the specific activities currently routinely achieved in practice with [177Lu]Lu-PSMA-617 . Incorporation of high loads of Lutetium-177 represents a critical parameter to obtain a product bearing therapeutic clinical doses of radioactivity (i.e., in the range of 200–400 mCi) in relatively low amounts of precursor, thus avoiding the need of injecting ligand doses that would saturate tumor structures . After radiolabelling, [177Lu]Lu-OncoFAP-DOTAGA is highly stable and presents high affinity towards isolated hFAP and against FAP-positive cancer cells.
As an alternative to 68Ga for diagnostic applications, we also developed two novel OncoFAP-conjugates bearing NODAGA and NOTA radiometal chelators, which are known to form stable complexes with Al18F [27–31]. We performed the radiochemical synthesis of both [18F]AlF-OncoFAP-NODAGA and [18F]AlF-OncoFAP-NOTA. The radiolabelling of OncoFAP-NODAGA with [18F]AlF resulted in very low radiochemical yields and radiochemical purities, probably due to the possibility of the NODAGA chelator to form a stable neutral complex with the Al3+ ion, thus partially preventing the incorporation of the fluoride (18F−) ion. On the contrary, OncoFAP-NOTA, which devoids a carboxylic acid function, was efficiently radiolabelled with [18F]AlF, with excellent purity and a radiochemical yield of approximately 20%. [18F]AlF-OncoFAP-NOTA retains affinity towards hFAP (co-elution experiments) and FAP-positive SK-RC-52 cells showing binding proprieties similar to the ones of [68Ga]Ga-OncoFAP-DOTAGA. In vivo biodistribution studies in tumor-bearing mice confirmed favourable tumor-targeting performance of [18F]AlF-OncoFAP-NOTA. The novel radiotracer selectively accumulates in solid lesion, with high tumour-to-background ratio at early time points (i.e., 1 hour after systemic administration), thus supporting further development of the molecule as diagnostic PET radiopharmaceutical in clinical practice. The biodistribution profile of [18F]AlF-OncoFAP-NOTA in murine model is comparable with the one of the clinically validated [68Ga]Ga-OncoFAP-DOTAGA . Application of [18F]AlF-OncoFAP-NOTA as a PET/CT imaging agent may result in significant logistical and clinical advantages as a consequence of the imaging characteristic of the isotope. Fluorine-18 is characterized by ∼97% β+ emission, 635 keV maximum positron energy, short β+ trajectory with mean positron range of 0.27 mm in soft tissue. Moreover, it can be produced in large scale in cyclotrons [32, 33] with the possibility of a subsequent delivery to satellite sites other than the production site, as the half-life of the isotope is long enough to allow this strategy (t1/2 = 109.8 min). Translation in the clinical setting will be of significant impact also in terms of daily organization of the PET/CT schedule. In particular, the use of 18F-based imaging procedures allows an easier patient preparation (no need of fasting, no need of maintaining low glucose level) and offer an optimal imaging time window of approximately 30–180 min after tracer injection. In addition, OncoFAP-derivatives can be exploited as PET/CT imaging agents for a broader window of malignancies, which are not efficiently detected by other marketed radiopharmaceuticals, such as [18F]FDG, [18F]FLT, [18F]F-MCH and [18F]F-PSMA-1007 [1, 34]. Examples are represented by esophageal , liver  and pancreatic cancers , brain primary tumours and metastases  or head and neck cancers .
Our results provide the reference for robust and efficient radiolabelling methodologies of OncoFAP-radioligand products that are easy to implement in clinical practice in small, medium and large size hospital radiopharmacies. The different radiosynthetic strategies presented in this article for each radioisotope can be selected on the basis of the specific needs of the various clinical centers. Automated production of [68Ga]Ga-OncoFAP-DOTAGA, a clinically validated PET tracer , and of [177Lu]Lu-OncoFAP-DOTAGA will facilitate the implementation of this theranostic pair in clinical practice. Efficient production of [18F]AlF-OncoFAP-NOTA represents the basis for the implementation of this novel radiotracer for PET imaging applications.