Adoptive chimeric antigen receptor (CAR)-T cell therapies have led to tremendous clinical success, especially in treatment of hematological malignancies. However, optimizations are still required to tackle remaining challenges such as antigen escape, on-target off-tumor toxicity, limited availability of tumor-specific antigens as well as insufficient T cell persistence and tumor infiltration. A significant level of expectation is currently placed in transgenic T cell receptors (TCRs), which feature a highly sensitive, naturally evolved signaling machinery and cover a broader range of targets. This work aimed at combining both technologies, CAR and transgenic TCR, to increase the anti-tumor response, to facilitate dual-targeting, to minimize the risk for antigen escape and to potentially achieve synergistic effects. As proof of concept, initial experiments were focused on the combination of the clinically well-established second-generation CD20-CAR and the previously published transgenic dNPM1-TCR. CAR’TCR-T cells, co-expressing CD20-CAR and dNPM1-TCR, revealed increased anti-tumor activity upon dual stimulation, not only upon short-term co-culture but also upon long-term repetitive in vitro stimulation. Accordingly, CAR’TCR-T cells displayed a unique transcriptomic signature, indicating increased T cell activation and proximal signaling. In-depth characterization of CAR’TCR-T cell functionality also included the analysis of alternative costimulatory domains in the CAR designs, displaying no significant difference between 4-1BB- and CD28-costimulated CAR’TCR-T cells. Moreover, it was addressed whether co-expression of CD20-CAR promotes functional recovery of the MHC class I-restricted dNPM1-TCR in CD4+ T cells. Finally, dual-specificity and enhanced anti-tumor activity of CAR’TCR-T cells were verified in vitro and in vivo for a clinically relevant AML setting by co-expressing dNPM1-TCR and CD33-CAR. Especially the treatment with Triple-T cells, meaning a cellular composition of CAR’TCR-T, CAR-T and TCR-T, led to promising results in vivo, demonstrating increased anti-tumor cytotoxicity compared to a mixture of CAR-T and TCR-T cells. In summary, this work supports the approach of co-expressing a CAR and a transgenic TCR to achieve dual-specificity and enhanced T cell anti-tumor activity. The superior performance of Triple-T highlights the therapeutic benefit of CAR’TCR-T cells and the potential clinical applicability through co-transduction with two different lentiviral vectors.
