P-08.06 Optimizing promoter systems to enhance cytokine response in CAR T cells for tumor therapy

<h3>Background</h3> Chimeric antigen receptor T-cells (CAR-T) frequently face substantial challenges within the immunosuppressive tumor microenvironment (TME). To overcome these, fourth-generation CAR-T cells, known as TRUCKs, have been engineered to secrete immunomodulatory cytokines (e.g., IL-12) sometimes upon activation, thereby amplifying the local T cell response in an auto- and paracrine manner. Activation-induced secretion depends on activation-specific promoter systems, such as the NFAT6 promoter. However, NFAT6 reaches maximal activity only after 24 hours, delaying the timely release of cytokines needed to effectively modulate the TME.The primary goal of this study is to optimize activation-induced cytokine release in CAR-T by optimized promoter activity, hypothesizing that modified promoters will achieve earlier and higher cytokine release following CAR-engagement. <h3>Materials and Methods</h3> A luciferase reporter CAR-TRUCK is used for rapid screening of promoter activity in vitro. The optimized promoters will then be evaluated for impact of cytokine release, proliferation, survival, and cytotoxicity of the respective TRUCKs. Experiments are conducted in a fully murine system to enable the development of a mouse model for CAR-T therapy. Target gene activation was assessed via a luciferase reporter in murine T cells and in murine T cell lymphoma EL4 in vitro. <h3>Results</h3> We saw CAR signal in all the designed constructs (IL-2 promotor, CD69 promotor, self-designed NR4A1 models) in the packaging cell line as well as in target cells. Luciferase activity was relatively low in CD69, NFAT6, and IL-2 promotor system was highest in the self-designed NR4A1 model. Interestingly, an enhancer on the genomic level further upregulated the promotor activity. Activation was achieved with ionomycin. Further activators and optimal activation conditions are being evaluated to determine quickest and highest on as well as rapid off rates of the respective promoter systems. <h3>Conclusions</h3> While further experiments are required to validate our findings and draw definitive conclusions, we have first supporting data that a potent promoter will greatly enhance murine TRUCKs which we aim to transfer from the current blood cancer model to solid tumors, potentially paving the way for broader immunologic <i>in vivo</i> applications. L. Steger: None. F. Gsottberger: None. F. Müller: None. A. Mackensen: None. S. Petkovic: None. C. Büttner: None.