During the last years, immune-modulating drugs became an important cornerstone in the treatment of cancer patients. In particular, the PD1/PDL-1 and CTLA-4 antagonists revolutionized the field. However, only a limited number of patients benefit from these antagonistic molecules and more combination therapies are on the way to increase the number of patients benefiting from these novel therapies. Among the combinations, drugs that are T-cell or myeloid cell agonists belonging to the TNFR-superfamily show first promising clinical results. However, systemic immune activation bears the risk of severe side effects that will not allow using these powerful drugs at an effective dose. We have developed a new class of DARPin molecules that enable tumor-restricted immune cell activation of TNFR-superfamily agonists in the tumor only, thereby preventing systemic immune-activation. We generated DARPin molecules that bind with high affinity to TNFR-superfamily members (CD134, CD137 and CD40) and DARPin molecules that bind to tumor-specific antigens such as HER2 and EGFR or targets restricted to the tumor stroma compartment like FAP and extra-domain B of fibronectin (ED-B). Using these building blocks from our DARPin toolbox, we constructed a variety of multi-specific molecules consisting of a TNFR-superfamily receptor targeting DARPin molecule and a tumor-localizing DARPin molecule. In reporter cell assays the multi-specific DARPin molecules activate the respective TNF-superfamily receptor only in the presence of a cell expressing the tumor-localizer; e.g. the FAP-CD134 molecule activates CD134 only in the presence of a stromal cell expressing FAP and not in its absence. Moreover, activation of CD134 was dependent on the density of FAP expression on cells, showing that CD134 only becomes activated if a certain level of the tumor-localizing target is expressed. These finding were confirmed in experiments with primary immune cells where we see immune-cell activation only upon binding to the tumor-localizing target. This could be shown for multiple combinations (>8) and supports the concept that multi-specific DARPin molecules are powerful drug-candidates allowing tumor-restricted immune cell activation.

Citation Format: Ulrike Fiedler, Christian Reichen, Joanna Taylor, Patricia Schildknecht, Sophie Barsin, Clara Metz, Anja Schlegel, Simon Fontaine, Denis Villemagne, Julia Ahlskog, Yvonne Kaufmann, Alexander Link, Nicolo Rigamonti, Julia Hepp, Michael T. Stumpp. Tumor-restricted immune modulation by multispecific molecules from the DARPin toolbox [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4552.