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.Small organic ligands which selectively bind with high affinity to tumor-associated antigens are increasingly applied as targeting delivery vehicles of small payloads such as radionuclides (
1,
2), drugs (
3–
5), and fluorophores (
6,
7) to tumor sites. In principle, the use of small ligands for targeting applications offers several advantages compared to intact immunoglobulins, including superior penetration of solid neoplastic lesions (
8), lower immunogenicity (
9), and a reduced cost of goods (
10). Low molecular weight compounds may reach their target in vivo in a matter of seconds, thanks to rapid extravasation after intravenous administration (
8). A strikingly selective accumulation of small ligands in neoplastic masses has been demonstrated for a small number of targets including somatostatin receptor type 2 (SSTR-2) (
11), prostate-specific membrane antigen (PSMA) (
12), and carbonic anhydrase IX (CAIX) (
13), for which high-affinity small organic ligands are available. Those ligands are typically specific for defined tumor entities, such as neuroendocrine tumors (
11), prostate cancer (
3), and clear cell renal cell carcinoma (
2).
177Lu-DOTATATE (Lutathera), a small-molecule product targeting SSTR-2, has been approved based on phase III data in which a clinically meaningful 82% reduction in the risk of disease progression or death was demonstrated in patients with gastroenteropancreatic neuroendocrine tumors (GEP-NETs) (
14). Similar data are expected from the currently ongoing phase III VISION trial for
177Lu-PSMA-617 (clinical trial no.
{"type":"clinical-trial","attrs":{"text":"NCT03511664","term_id":"NCT03511664"}}NCT03511664), a radiolabeled small molecule that binds with high affinity to PSMA and that enables targeted beta particle therapy in metastatic castration-resistant prostate cancer patients (
15). PHC-102, a
99mTc-labeled small-molecule derivative targeting CAIX, exhibited favorable uptake in primary and metastatic lesions in patients with renal cell carcinoma (RCC) (
2). In light of the promising performance of small organic ligands, it would be desirable to discover and develop small molecules with a broader tumor-targeting potential, therefore covering multiple cancer types.Fibroblast activation protein (FAP) is a type II integral membrane serine protease which is abundantly expressed in the stroma of more than 90% of the epithelial cancers, including malignant breast, colorectal, skin, prostate, and pancreatic cancers (
16,
17), while exhibiting a restricted expression in normal adult tissues (
18,
19). Haberkorn and coworkers (
1,
20,
21) have recently described a series of FAP ligands capable of selective accumulation in FAP-positive tumors in mice and in patients. One of these products (named FAPI-04) showed impressive tumor to background ratios at early time points (i.e., few hours after administration) in a broad range of different cancer types in patients. More than 28 tumor types including breast, lung, pancreatic, head and neck, esophagus, and colorectal cancer presented a remarkably high uptake of a FAP-targeted small molecule labeled with gallium-68 (
1,
20,
21). For this reason, FAP has recently been dubbed as “the next billion-dollar target for theranostic products” (
22).Here, we describe how the chemical modification of a quinoline moiety in position 8 led to the discovery of OncoFAP, a small organic FAP ligand with a dissociation constant in the subnanomolar concentration range. OncoFAP exhibited a strikingly selective and efficient tumor-targeting performance when equipped with various types of payloads, including radionuclides, fluorophores, and cytotoxic drugs. The targeting delivery of radionuclides to solid tumors is rapidly gaining in popularity, as it may open theranostic opportunities, associated with the use of gallium-68 for positron emission tomography (PET) imaging and of lutetium-177 for therapeutic applications (
23). The delivery of fluorescein to tumors enables the conditional activation of chimeric antigen receptor (CAR) T cells, which display a potent biocidal activity only in the presence of fluorescein-labeled adaptor molecules specific to a tumor antigen (
24,
25). Finally, small-molecule–drug conjugates (SMDCs) promise to represent a valid alternative to antibody–drug conjugates for cancer therapy, with better tumor penetration and a lower cost of goods (
8,
26,
27).
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