Tumor-targeted delivery of Interleukin-2 enhances the anti-cancer activity of OncoFAP radioligand therapeutics

Introduction: Fibroblast Activation Protein (FAP) is a cell surface antigen abundantly expressed in the microenvironment of the majority of solid human malignancies. First clinical results of FAP targeting radioligand therapy with fibroblast activation protein inhibitor (FAPI) derivatives leave room for improvement of tumor retention time and efficacy. This may be achieved by the use of novel FAP ligands and/or combination therapies. Recently, we described successful clinical translation of the novel FAP ligand OncoFAP. Its bivalent derivate BiOncoFAP demonstrated long lasting accumulation in small animal tumor models. We here present first results from therapy of ¹⁷⁷Lu-OncoFAP and ¹⁷⁷Lu-BiOncoFAP in murine tumor models at submaximal doses in combination with L19-IL2, a clinical stage immunocytokine.

Methods: Female athymic 6-8 weeks old Balb/c AnNRj-Foxn1 mice were randomized into 7 groups of 7 mice each. All mice were implanted subcutaneously with 2 x 10⁶ of either HT-1080.hFAP or HT-1080.wt (wild type control) cells. 7 Days after implantation, mice were i.v. injected with saline or 5 MBq of Lu-177-OncoFAP/-BiOncoFAP. On days 8, 10 and 12 after tumor implantation, mice were s.c. injected with either saline or 2.5mg/kg bodyweight of L19-IL2, a fusion protein consisting of an antibody specific to the alternatively spliced extra-domain B of fibronectin and human interleukin-2. Mice weight and tumor growth were monitored daily, up to 35 days. In treated SK-RC-52.hFAP tumor models, Immunofluorescence and mass-spectrometry based proteomics analysis was performed for markers of natural killer cells.

Results: Control groups and monotherapy with ¹⁷⁷Lu-OncoFAP or L19-IL2 led to no complete tumor cures. ¹⁷⁷Lu-BiOncoFAP monotherapy resulted in complete tumor remission in 2/7 mice. Combination therapy with L19-IL2 effectively enhanced the therapeutic efficacy of OncoFAP-based radioligand therapeutics, with complete remissions in 6/7 mice for ¹⁷⁷Lu-OncoFAP, and in 7/7 mice for ¹⁷⁷Lu-BiOncoFAP (Figure 1). After combination treatment, immunofluorescence and proteomic studies revealed significant increase in presence of NKp46-positive cells in the tumor microenvironment with enhanced expression of pro-apoptotic enzymes.

Conclusions: The combination with an anti-EDB antibody-interleukin-2 fusion protein enhanced anti-tumor effect at submaximal therapeutic doses of FAP-targeted radioligand therapy based on OncoFAP-derivatives. Immunofluorescence and proteomic studies point toward a mechanism of action based on activation of natural killer cells.

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