Immunotherapy as an approach to cancer treatment began in the late 19th century when William B. Coley developed an early form of immunotherapy using a mixture of toxin-producing bacteria. In the project, we will revive Coley's original idea, but in a modernized version. Specifically, we will use genetic information taken from toxin-producing bacteria, which we will introduce locally to tumor tissue using a gene therapy approach called gene electrotransfer (GET). The selection of toxins for the project is based on what we believe is one of the most promising and simplest approaches to induce antitumor immunity, namely in situ vaccination. Various local ablative techniques can be used as in situ vaccines by releasing tumor antigens (TA) in the context of danger signals. However, this is usually not sufficient to elicit an effective systemic and durable immune response. Thus, these ablative therapies must be combined with immunological adjuvants, as is often the case with standard vaccines. Therefore, we plan to use a combination of two toxin-encoding genes in this project: The first is intended for in situ vaccination and the second to boost the primed immune response against the released TAs. The candidate genes for the first toxin will be chosen from the group of pore-forming toxins. These toxins are known to cause cell lysis and therefore can be used to release TAs from tumor cells. The second toxins will be selected from the group of immunostimulatory toxins, named superantigens (SAgs). These toxins can trigger polyclonal T cell expansion and massive cytokine release. As a result, SAgs can alter the microenvironment and amplify the specific immune response against TAs released by tumor cells at the time of exposure. In the project, we plan to use GET to deliver plasmids encoding a secretory form of toxins. GET allows direct transfection into the tumor and only into the tumor, which would lead to transient transfection and paracrine secretion of the expressed toxin. Therefore, a single transfection of only a few cells in the tumor should be sufficient to achieve a therapeutic effect.
Taken together, the objective of the project is to prepare new toxin-encoding plasmids for use in GET to induce in situ vaccination. Since this is a completely new approach, a large part of the project will be dedicated to plasmid design and testing of plasmid functionality. In doing so, we will first select the most suitable toxin-encoding genes and then design plasmids that will allow their secretion from transfected cells. Next, we will investigate whether toxins produced and secreted by eukaryotic cells instead of bacteria retain their toxic and superantigenic activity. Finally, the GET of newly constructed plasmids will be tested in vivo to determine their safety and antitumor efficacy in mouse tumor models.