Cancer remains one of the most pressing health problems worldwide warranting the continued development of new cancer treatments. Cytokines, such as IFN and TNF, hold great promise, as they are endogenous mediators of cancer surveillance. However, systemic toxicity prevents their full clinical application. In this regard, AcTakines (Activated-by-Targeting Cytokines) are being developed. They resemble immunocytokines as they are composed of cytokines fused to cellspecific targeting domains, but the cytokines harbor mutations that strongly reduce binding to their receptor complex. As such, AcTakines are inactive while traveling through the body and only unveil their activity upon cell-specific binding, leading to an up to 1000-fold enhanced activity only on the targeted cells. In this project, we aim to evaluate the in vivo use of AcTakines as antitumor agents. Having obtained proof-of-concept, we aim to target TNF or IFN cytokine activity directly to the tumor, tumor neovasculature and/or infiltrating immune cells of different tumor models. Thorough analysis of the tumor niche, i.e. composition and accessibility, will allow optimizing treatment modalities. The in situ effect of different AcTakines will be monitored. Finally, differently targeted AcTakines as single agents or in combinations will be evaluated. Defining optimized AcTakine treatment regimens in these preclinical settings will be of help to advance this innovative class of biologics towards the clinic.