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.

Glycosylation of proteins profoundly impacts their physical and biological properties. Yet our ability to engineer novel glycoprotein structures remains limited. Established bacterial glycoengineering platforms require secretion of the acceptor protein to the periplasmic space and preassembly of the oligosaccharide substrate as a lipid-linked precursor, limiting access to protein and glycan substrates respectively. Here, we circumvent these bottlenecks by developing a facile glycoengineering platform that operates in the bacterial cytoplasm. The Glycoli platform leverages a recently discovered site-specific polypeptide glycosyltransferase together with variable glycosyltransferase modules to synthesize defined glycans, of bacterial or mammalian origin, directly onto recombinant proteins in the E. coli cytoplasm. We exploit the cytoplasmic localization of this glycoengineering platform to generate a variety of multivalent glycostructures, including self-assembling nanomaterials bearing hundreds of copies of the glycan epitope. This work establishes cytoplasmic glycoengineering as a powerful platform for producing glycoprotein structures with diverse future biomedical applications.

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.

Engineered cytokines are gaining importance in cancer therapy, but these products are often limited by toxicity, especially at early time points after intravenous administration. 4-1BB is a member of the tumor necrosis factor receptor superfamily, which has been considered as a target for therapeutic strategies with agonistic antibodies or using its cognate cytokine ligand, 4-1BBL. Here we describe the engineering of an antibody fusion protein, termed F8-4-1BBL, that does not exhibit cytokine activity in solution but regains biological activity on antigen binding. F8-4-1BBL bound specifically to its cognate antigen, the alternatively spliced EDA domain of fibronectin, and selectively localized to tumors in vivo, as evidenced by quantitative biodistribution experiments. The product promoted a potent antitumor activity in various mouse models of cancer without apparent toxicity at the doses used. F8-4-1BBL represents a prototype for antibody-cytokine fusion proteins, which conditionally display “activity on demand” properties at the site of disease on antigen binding and reduce toxicity to normal tissues.