Immunotherapeutic strategies are becoming increasingly widespread in the fight against cancer. One such approach is T cell based therapy with lymphocytes T, expressing chimeric antigen receptor (CAR) on their surface. CARs are synthetic receptors composed of multiple molecular components, typically derived from various elements of the immune system. CAR T cell therapy has proven effective in treating certain hematological malignancies, leading to the approval of several cellular products for clinical use in patients who are unresponsive to other forms of treatment or experience relapse. However, the broader use of CAR T cell therapy is limited by significant side effects and other challenges associated with the therapy. To enhance and optimize CAR T cell therapy, various options for improving the CAR construct and its domain are being explored.
In this doctoral dissertation, we developed and tested new CAR constructs incorporating co-stimulatory domains from the innate immune system's Toll-like receptor (TLR) signaling pathway. Our primary design focused on a CAR targeting the CD19 antigen, with a 4-1BB co-stimulatory domain and a CD3ζ activation domain. Based on initial screening experiments, we selected for further testing CAR constructs with a truncated TLR4 co-stimulatory domain, a construct with the myeloid differentiation primary response protein (MyD88), and a construct with a truncated adapter containing the TIR-domain-containing adapter-inducing interferon-β (TRIF). We generated CAR T cells using lentiviral transduction and observed significantly enhanced activation, indicated by cytokine IL-2 release, when co-cultured with CD19-positive hematological malignant cells and solid tumor cells. Certain selected CAR T designs also exhibited improved cytotoxicity and increased release of INF-γ. Notably, we did not detect non-specific cytokine release, suggesting an absence of substantial tonic signaling. Analysis of cellular immunological markers in the absence of antigen revealed no significant phenotypic differences between the various CAR T cells. RNA sequencing analysis indicated increased expression of specific genes involved in the innate immune response, T cell activation, and proliferation. In xenograft mouse models with CD19+ solid tumors, CAR T cells with MyD88 and TLR4 exhibited prolonged remission compared to the conventional CAR T cells. Conversely, experiments with CAR TRIF showed reduced efficacy and characteristics that might limit its functionality. This doctoral thesis demonstrates the potential of TLR activation domains in CAR T cell therapy while highlighting that the inclusion of some signaling pathways may reduce the long-term efficacy of CAR T cells.
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