We describe the development of OncoFAP, an ultra-high-affinity ligand of fibroblast activation protein (FAP) for targeting applications with pan-tumoral potential. OncoFAP binds to human FAP with affinity in the subnanomolar concentration range and cross-reacts with the murine isoform of the protein. We generated various fluorescent and radiolabeled derivatives of OncoFAP in order to study biodistribution properties and tumor-targeting performance in preclinical models. Fluorescent derivatives selectively localized in FAP-positive tumors implanted in nude mice with a rapid and homogeneous penetration within the neoplastic tissue. Quantitative in vivo biodistribution studies with a lutetium-177–labeled derivative of OncoFAP revealed a preferential localization in tumors at doses of up to 1,000 nmol/kg. More than 30% of the injected dose had already accumulated in 1 g of tumor 10 min after intravenous injection and persisted for at least 3 h with excellent tumor-to-organ ratios. OncoFAP also served as a modular component for the generation of nonradioactive therapeutic products. A fluorescein conjugate mediated a potent and FAP-dependent tumor cell killing activity in combination with chimeric antigen receptor (CAR) T cells specific to fluorescein. Similarly, a conjugate of OncoFAP with the monomethyl auristatin E-based Vedotin payload was well tolerated and cured tumor-bearing mice in combination with a clinical-stage antibody-interleukin-2 fusion. Collectively, these data support the development of OncoFAP-based products for tumor-targeting applications in patients with cancer.

Purpose The fibroblast activation protein (FAP) is an emerging target for molecular imaging and therapy in cancer. OncoFAP is a novel small organic ligand for FAP with very high affinity. In this translational study, we establish [68Ga]Ga-OncoFAP-DOTAGA (68Ga-OncoFAP) radiolabeling, benchmark its properties in preclinical imaging, and evaluate its application in clinical PET scanning. Methods 68Ga-OncoFAP was synthesized in a cassette-based fully automated labeling module. Lipophilicity, affinity, and serum stability of 68Ga-OncoFAP were assessed by determining logD7.4, IC50 values, and radiochemical purity. 68Ga-OncoFAP tumor uptake and imaging properties were assessed in preclinical dynamic PET/MRI in murine subcutaneous tumor models. Finally, biodistribution and uptake in a variety of tumor types were analyzed in 12 patients based on individual clinical indications that received 163 ± 50 MBq 68Ga-OncoFAP combined with PET/CT and PET/MRI. Results 68Ga-OncoFAP radiosynthesis was accomplished with high radiochemical yields. Affinity for FAP, lipophilicity, and stability of 68Ga-OncoFAP measured are ideally suited for PET imaging. PET and gamma counting–based biodistribution demonstrated beneficial tracer kinetics and high uptake in murine FAP-expressing tumor models with high tumor-to-blood ratios of 8.6 ± 5.1 at 1 h and 38.1 ± 33.1 at 3 h p.i. Clinical 68Ga-OncoFAP-PET/CT and PET/MRI demonstrated favorable biodistribution and kinetics with high and reliable uptake in primary cancers (SUVmax 12.3 ± 2.3), lymph nodes (SUVmax 9.7 ± 8.3), and distant metastases (SUVmax up to 20.0). Conclusion Favorable radiochemical properties, rapid clearance from organs and soft tissues, and intense tumor uptake validate 68Ga-OncoFAP as a powerful alternative to currently available FAP tracers.

Imaging procedures based on small molecule-radio conjugates (SMRCs) targeting fibroblast activation protein (FAP) have recently emerged as a powerful tool for the diagnosis of a wide variety of tumours. However, the therapeutic potential of radiolabeled FAP-targeting agents is limited by their short residence time in neoplastic lesions. In this work, we present the development and in vivo characterization of BiOncoFAP, a new dimeric FAP-binding motif with extended tumour residence time and favorable tumour-to-organ ratio. Methods: The binding properties of BiOncoFAP and its monovalent OncoFAP analogue were assayed against recombinant hFAP. Preclinical experiments with [ 177 Lu]Lu-OncoFAP-DOTAGA ( 177 Lu-OncoFAP) and [ 177 Lu]Lu-BiOncoFAP-DOTAGA ( 177 Lu-BiOncoFAP) were performed in mice bearing FAP-positive HT-1080 tumours.Results: OncoFAP and BiOncoFAP displayed comparable sub-nanomolar dissociation constants towards hFAP in solution, but the bivalent BiOncoFAP bound more avidly to the target immobilized on solid supports. In a comparative biodistribution study, 177 Lu-BiOncoFAP exhibited a more stable and prolonged tumour uptake than 177 Lu-OncoFAP (~20% ID/g vs ~4% ID/g, at 24h p.i., respectively). Notably, 177 Lu-BiOncoFAP showed favorable tumour-to-organ ratios with low kidney uptake. Both 177 Lu-OncoFAP and 177 Lu-BiOncoFAP displayed potent anti-tumour efficacy when administered at therapeutic doses in tumour bearing mice. Conclusions:177 Lu-BiOncoFAP is a promising candidate for radioligand therapy of cancer, with favorable in vivo tumour-to-organ ratio, long tumour residence time and potent anti-cancer efficacy.

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Starting from the structure of previously reported 3-Br-isoxazoline-based covalent inhibitors of P. falciparum glyceraldehyde 3-phosphate dehydrogenase, and with the intent to improve their metabolic stability and antimalarial activity, we designed and synthesized a series of simplified analogues that are characterized by the insertion of the oxadiazole ring as a bioisosteric replacement for the metabolically labile ester/amide function. We then further replaced the oxadiazole ring with a series of five-membered heterocycles and finally combined the most promising structural features. All the new derivatives were tested in vitro for antimalarial as well as antileishmanial activity. We identified two very promising new lead compounds, endowed with submicromolar antileishmanial activity and nanomolar antiplasmodial activity, respectively, and a very high selectivity index with respect to mammalian cells.

Imaging procedures based on small molecule-radio conjugates (SMRCs) targeting fibroblast activation protein (FAP) have recently emerged as a powerful tool for the diagnosis of a wide variety of tumours. However, the therapeutic potential of radiolabeled FAP-targeting agents is limited by their short residence time in neoplastic lesions. In this work, we present the development and in vivo characterization of BiOncoFAP, a new dimeric FAP-binding motif with extended tumour residence time and favorable tumour-to-organ ratio. Methods: The binding properties of BiOncoFAP and its monovalent OncoFAP analogue were assayed against recombinant hFAP. Preclinical experiments with [177Lu]Lu-OncoFAP-DOTAGA (177Lu-OncoFAP) and [177Lu]Lu-BiOncoFAP-DOTAGA (177Lu-BiOncoFAP) were performed in mice bearing FAP-positive HT-1080 tumours. OncoFAP and BiOncoFAP displayed comparable sub-nanomolar dissociation constants towards hFAP in solution, but the bivalent BiOncoFAP bound more avidly to the target immobilized on solid supports. In a comparative biodistribution study, 177Lu-BiOncoFAP exhibited a more stable and prolonged tumour uptake than 177Lu-OncoFAP (~20% ID/g vs ~4% ID/g, at 24h p.i., respectively). Notably, 177Lu-BiOncoFAP showed favorable tumour-to-organ ratios with low kidney uptake. Both 177Lu-OncoFAP and 177Lu-BiOncoFAP displayed potent anti-tumour efficacy when administered at therapeutic doses in tumour bearing mice. 177Lu-BiOncoFAP is a promising candidate for radioligand therapy of cancer, with favorable in vivo tumour-to-organ ratio, long tumour residence time and potent anti-cancer efficacy.

Purpose: Small Molecule-Drug Conjugates (SMDCs) are modular anti-cancer pro-drugs that include a tumor-targeting small organic ligand, a cleavable linker and a potent cytotoxic agent. Most of the SMDC products that have been developed for clinical applications target internalizing tumor-associated antigens on the surface of tumor cells. We have recently described a novel non-internalizing small organic ligand (named OncoFAP) of Fibroblast Activation Protein (FAP), a tumor-associated antigen highly expressed in the stroma of most of solid human malignancies. Experimental Design: In this article, we describe a new series of OncoFAP-Drug derivatives based on monomethyl auristatin E (MMAE, a potent cytotoxic tubulin poison) and dipeptide linkers that are selectively cleaved by FAP in the tumor microenvironment. Results: The tumor targeting potential of OncoFAP was confirmed in cancer patients using Nuclear Medicine procedures. We used mass spectrometry methodologies in order to quantify the amount of pro-drug delivered to tumors and normal organs, as well as the efficiency of the drug release process. Linkers previously exploited for anticancer drug conjugates were used as benchmark. We identified OncoFAP-Gly-Pro-MMAE as the best performing SMDC, which has now been prioritized for further clinical development. OncoFAP-Gly-Pro-MMAE selectively delivered more than 10% injected dose per gram of MMAE to FAP-positive tumors, with a tumor-to-kidney ratio of 16:1 at 24-hours post-injection. Conclusions: The FAP-specific drug conjugates described in this article promise to be efficacious for the targeting of human malignancies. The extracellular release of potent anticancer payloads mediates durable complete remission in difficult-to-treat animal models of cancer.

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