A preclinical model for the identification of therapeutically active transgenes in local cancer immunotherapy

The efficacy of systemic immunotherapies is limited for poorly immunogenic tumors which suppress T cell priming and tumor infiltration. Modern antigen carriers including viral vectors and messenger RNA/lipid nanoparticle (LNP) combinations have renewed the interest in local immunotherapy due to their ability to express multiple transgene constructs simultaneously. The identification of therapeutically active combinations, however, is hampered by the lack of preclinical models to rapidly express and evaluate transgenes combinations <i>in vivo</i>. To enable empirical testing of immunogenic transgenes, we have combined a doxycycline-inducible expression system with flow cytometry and multiplex immunohistochemistry imaging. In animal models of liver and colon cancer, we demonstrate that the impact of a single transgene on the immune milieu is limited and heavily dependent on the studied tumor entity. Compared to single transgenes, transgene combinations induced more complex and only partially predictable alterations in the tumor micromilieu but strongly enhanced therapeutic efficacy. By combining expression of transgenes with impact on antigen-presenting cells and T cells, we identified a combination of IL-12, CXCL9 and FLT3L as the most promising combinatorial approach, resulting in complete tumor remissions in mice. Taken together, we demonstrate the ability of our preclinical model to identify therapeutic transgene combinations for more efficacious locoregional immunotherapy of solid tumors.