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The immunosuppressive tumor microenvironment is a major hurdle for cancer therapy. Our lab previously developed a cell and gene therapy strategy for the tumor-targeted delivery of IFN-alpha based on the transduction of hematopoietic stem/progenitor cells (HSPC) with lentiviral vectors (LV) expressing the IFN-alpha gene under the control of the TIE2 enhancer/promoter. The specificity of this strategy is ensured by the restricted expression of IFN-alpha in a population of HSPC-derived tumor-infiltrating macrophages expressing the TIE2 receptor (TEM), which are recruited at the tumor site. TEM-mediated delivery of IFN-alpha has shown efficacy in different tumor models of both solid and hematopoietic origin, including a model of human glioma in immunodeficient mice. New data obtained in a more relevant immunocompetent mouse model of orthotopic glioma closely reproducing several features of the human disease show dramatic tumor inhibition in mice treated with IFN-alpha gene therapy. We are currently characterizing the contribution of IFN-induced antiangiogenic and modulatory activity on the innate and adaptive immune system underlying such response. To further improve the therapeutic index of our strategy, we are developing inducible strategies to superimpose a timer and a rheostatic switch on the amount of IFN-alpha secreted in the tumor microenvironment. Indeed, whereas a sustained cytokine output could ensure efficacy and long-term protection from tumor recurrence, it may raise concerns for long-term effects, especially in case of cancer eradication. By fusing a destabilizing domain (DD) to a protein of interest (POI), the former can confer its instability to the latter. This destabilization can be rescued in a reversible and dose-dependent manner with the addition of a small molecule specifically binding to the DD. To apply this technology to our strategy, we have designed and in vitro tested different fusion proteins of IFN-alpha (DD-IFN-alpha) with or without the addition of flexible or cleavable linkers and selected them for their capacity to be stabilized in presence of their specific ligand in vitro. Through this approach, we have identified fusion proteins with low basal activity and high fold induction upon ligand treatment. These novel regulated forms of IFN-alpha are functional and their specific activities are comparable to the wild type. Based on preliminary evidence of in vivo ligand-regulated IFN release, we are now testing the safety and efficacy of our new platforms in inducing antitumor responses in melanoma, colon, and glioma models of cancer. This abstract is also being presented as Poster B31. Citation Format: Filippo Birocchi, Melania Cusimano, Anna Ranghetti, Tiziano Di Tomaso, Barbara Costa, Peter Angel, Nadia Coltella, Luigi Naldini. Development of chimeric forms of IFN-alpha for “on demand” in vivo cancer gene therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR13.