Significance: Fibroblast activation protein (FAP) has recently emerged as a tumor-associated antigen with abundant and selective expression in the majority of human solid malignancies. To the best of our knowledge, OncoFAP is the highest-affinity small organic FAP ligand reported to date, with a dissociation constant of 680 pM, as measured by fluorescence polarization. Upon intravenous administration, both fluorescent and radiolabeled OncoFAP derivatives exhibited a rapid and selective accumulation in FAP-positive tumors, sparing normal tissues. OncoFAP was also used as a modular component for the generation of therapeutic products, enabling the targeted delivery of a potent beta-emitter (lutetium-177), of fluorescein-specific chimeric antigen receptor (CAR) T cells or of highly cytotoxic auristatin derivatives to FAP-positive tumors in vitro and in vivo.

Purpose: Antibody–drug conjugates and small-molecule-drug conjugates have been proposed as alternatives to conventional anticancer cytotoxic agents, with the potential to deliver bioactive payloads to the site of disease, helping spare normal tissues. Experimental Design: Here, we describe a novel small-molecule-drug conjugate, based on a high-affinity ligand specific to carbonic anhydrase IX. The product featured a peptidic linker, suitable for cleavage in the tumor extracellular environment, and monomethyl auristatin E as cytotoxic payload. Results: A potent anticancer activity was observed in nude mice bearing SKRC-52 renal cell carcinoma xenografts, but no durable complete responses could be observed in this model. However, when the product was administered together with L19-IL2 (a clinical-stage fusion protein capable of delivering IL2 to the tumor neovasculature), all treated mice in the combination group could be rendered tumor free, in a process that favored the influx of natural killer cells into the tumor mass. The combination of L19-IL2 and the new small-molecule-drug conjugate also eradicated cancer in 100% of immunocompetent mice, bearing subcutaneously grafted CT26 colorectal cancer cells, which stably expressed carbonic anhydrase IX. Conclusions: These findings may be of clinical significance, because carbonic anhydrase IX is overexpressed in the majority of clear cell renal cell carcinomas and in approximately 30% of colorectal cancers. The targeted delivery of IL2 helps potentiate the action of targeted cytotoxics, leading to cancer eradication in models that cannot be cured by conventional chemotherapy. Clin Cancer Res; 24(15); 3656–67. ©2018 AACR.

Imaging procedures based on small-molecule radioconjugates targeting fibroblast activation protein (FAP) have recently emerged as a powerful tool for the diagnosis of a wide variety of tumors. 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 an extended tumor residence time and favorable tumor-to-organ ratio. Methods: The binding properties of BiOncoFAP and its monovalent OncoFAP analog were assayed against recombinant human FAP. Preclinical experiments with ¹⁷⁷Lu-OncoFAP-DOTAGA (¹⁷⁷Lu-OncoFAP) and ¹⁷⁷Lu-BiOncoFAP-DOTAGA (¹⁷⁷Lu-BiOncoFAP) were performed on mice bearing FAP-positive HT-1080 tumors. Results: OncoFAP and BiOncoFAP displayed comparable subnanomolar dissociation constants toward recombinant human FAP 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 tumor uptake than ¹⁷⁷Lu-OncoFAP (∼20 vs. ∼4 percentage injected dose/g, respectively, at 24 h after injection). Notably, ¹⁷⁷Lu-BiOncoFAP showed favorable tumor-to-organ ratios with low kidney uptake. Both ¹⁷⁷Lu-OncoFAP and ¹⁷⁷Lu-BiOncoFAP displayed potent antitumor efficacy when administered at therapeutic doses to tumor-bearing mice. Conclusion:¹⁷⁷Lu-BiOncoFAP is a promising candidate for radioligand therapy of cancer, with favorable in vivo tumor-to-organ ratios, a long tumor residence time, and potent anticancer efficacy. Visual Abstract