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.

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christian pellegrino & Dario neri * engineered cytokine products represent a growing class of therapeutic proteins which need to be tested for biological activity at various stages of pharmaceutical development. in most cases, dedicated biological assays are established for different products, in a process that can be time-consuming and cumbersome. Here we describe the development and implementation of a universal cell-based reporter system for various classes of immunomodulatory proteins. the novel system capitalizes on the fact that the signaling of various types of pro-inflammatory agents (e.g., cytokines, chemokines, Toll-like receptor agonists) may involve transcriptional activation by NF-κB. Using viral transduction, we generated stably-transformed cell lines of B or T lymphocyte origin and compared the new reporter cell lines with conventional bioassays. The experimental findings with various interleukins and with members of the TNF superfamily revealed that the newly-developed "universal" bioassay method yielded bioactivity data which were comparable to the ones obtained with dedicated conventional methods. The engineered cell lines with reporters for NF-κB were tested with several antibody-cytokine fusions and may be generally useful for the characterization of novel immunomodulatory products. the newly developed methodology also revealed a mechanism for cytokine potentiation, based on the antibody-mediated clustering of TNF superfamily members on tumor-associated extracellular matrix components.