P808: controlled fratricide to augment anti-myeloma reactivity of slamf7 and cd38 car t cells

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: SLAMF7 and CD38 are expressed on multiple myeloma cells, but are also present on T cell subsets. The expression of a SLAMF7- or CD38-specific CAR therefore leads to mutual recognition of the T cells. Depending on the antigen expression level and kinetic, this results in varying degrees of fratricide, limited expansion rates and premature exhaustion. SLAMF7 and CD38 expression is upregulated after T cell activation, hampering the production of sufficient quantities of SLAMF7 CAR T cells and making the production of CD38 CAR T cells virtually impossible. Aims: We aim to prevent fratricide and augment the yield and fitness of SLAMF7 CAR T cells. We further aim to enable CD38 CAR T cell production, which is almost impossible due to the high CD38 expression on activated T cells and rampant fratricide. Instead of using gene-editing strategies, which are currently still lacking precision and need individual adaptions for each target antigen, we aim for an universal and easy to handle approach using the protein-kinase inhibitor Dasatinib. Dasatinib is readily available, affordable and can be removed from the culturing medium by washing steps. Methods: First, we determined the expression kinetics of the target antigens during the manufacturing by flow cytometry and afterwards tested different concentrations of Dasatinib during different stages of SLAMF7 and CD38 CAR T cell production. This way the optimal balance between fratricide prevention and the enrichment of CAR-expressing cells due to mutual stimulation was determined. The final T cell yield, CAR expression rate and the fitness of the cells were evaluated by flow cytometry, functional assays and RNA expression profiling. Results: We evaluated the kinetic of SLAMF7 and CD38 expression during CAR T cell manufacturing to predict the course of fratricide. The expression of both molecules rapidly increases after the activation of the cells. The expression of SLAMF7 peaks after 6 days and then decreases back to baseline levels within the next week, while CD38 stays continually expressed on the T cells during the next 2 weeks of cell culture. We tested different Dasatinib treatment schedules and concentrations during SLAMF7 CAR T cell production. In accordance with the SLAMF7 expression kinetic, the singular addition of 50 nM Dasatinib after gene transfer showed the most promising results, with an almost 3-fold increase in cell expansion of SLAMF7 CAR T cells. The percentage of cells, which were triple positive for the exhaustion markers TIM3, PD1 and LAG3 was reduced by half and the cells showed a less differentiated phenotype. For the successful manufacturing of CD38 CAR T cells, Dasatinib had to be administered during the complete manufacturing process to successfully inhibit fratricide and significantly increase cell expansion. After Dasatinib treatment, the CAR T cells expressed significant less exhaustion markers and were less differentiated compared to the untreated control cells. The CAR T cells showed full functionality after antigen-specific stimulation with target cells. Summary/Conclusion: By adding Dasatinib, we enable the production of CD38 CAR T cells, without diminishing anti-CD38 reactivity and without the need of gene editing. The yield of SLAMF7 CAR T cells can be significantly increased under Dasatinib treatment. These CAR T cells show a significantly better state of fitness, which could be reflected in the therapeutic success. Overall, we are presenting a strategy to expand the repertoire of myeloma target antigens and therefore increase patient access to CAR therapy. Keywords: CD38, CAR-T, Multiple myeloma