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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Antibody-based delivery of bioactive molecules represents a promising strategy for the improvement of cancer immunotherapy. Here, we describe the generation and characterization of R6N, a novel fully human antibody specific to the alternatively spliced domain D of Tenascin C, which is highly expressed in the stroma of primary tumors and metastasis. The R6N antibody recognized its cognate tumor-associated antigen with identical specificity in mouse and human specimens. Moreover, the antibody was able to selectively localize to solid tumors in vivo as evidenced by immunofluorescence-based biodistribution analysis. Encouraged by these results, we developed a novel fusion protein (termed mIL12-R6N) consisting of the murine interleukin 12 fused to the R6N antibody in homodimeric tandem single-chain variable fragment arrangement. mIL12-R6N exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. The experiments presented in this work provide a rationale for possible future applications for the R6N antibody for the treatment of cancer patients.

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.