AAV-mediated intra-tumoral immunotherapy for the treatment of immunologically “cold” tumors

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Recombinant adeno-associated virus vectors (AAV) represent a safe and efficient tool for in vivo gene therapy. Modification of the viral capsid enables generation of novel AAV variants suitable for in vivo intra-tumoral gene transfer, targeting both tumor cells and cells from the tumor microenvironment. Such vectors represent promising tools for in situ immunotherapy of tumors. The aim of the project is to manipulate the tumor and its microenvironment to increase tumor immunogenicity. For that, we will make use of engineered AAV to stimulate innate and adaptive immunity and to induce two types of immunogenic death (ICD), namely pyroptosis or necroptosis.In the first strategy, we will make use of AAV to express secreted or cytoplasmic flagellin. Flagellin is a potent bacterial-derived protein vaccine adjuvant sensed by Toll-like receptor-5 (TLR5) for example on macrophages/dendritic cells (DC). Sensing activates the innate immune system via the NF-kappa B pathway. Flagellin also induces pyroptosis through formation of the NLRC4 inflammasome. Pyroptosis is defined as a highly inflammatory variant of cell death and its key molecular executioner has recently been identified as gasdermin D (GSDMD), a downstream target of the inflammasome pathway. GSDMD cleavage by activated caspase-1 leads to pore formation and release of the active forms of the proinflammatory cytokines IL-1beta and IL-18, representing a catastrophic form of cell death, favoring the release of danger associated molecular patterns (DAMPs) as well as tumor antigens.Induction of ICD is emerging as potent trigger of anti-tumor immune responses and can be induced not only by the activation of pyroptosis executioners like GSDMD or gasdermin E (GSDME), but also by the activation of Mixed Lineage Kinase Domain-like Protein (MLKL), which can similarly oligomerize and also directly disrupts the membrane integrity to cause necroptosis. Therefore, in our second, complementary strategy, we will use AAV to induce ICD intra-tumorally by expressing active truncated/mutated forms of GSDMD, GSDME or MLKL, thus bypassing tumor resistance to the induction of pyroptosis or necroptosis. Induction of tumor cell death in the context of acute inflammation and release of DAMPs in conjunction with tumor antigens is expected to enhance presentation of tumor antigens via DC to T cells and finally induce a robust, long lasting adaptive anti-tumor immunity.Importantly, there is a strong scientific rationale for the combination of our two complementary approaches, since synergistic effects can be expected. In addition, our strategy can be combined easily with established immunotherapeutic approaches (e.g. immune checkpoints blockade). The favorable safety profile of AAV underscores the high potential for clinical translation of our strategy. Finally, our strategy offers the considerable potential to increase tumor immunogenicity even in immunologically “cold” tumors that still represent a clinical challenge.