Chimeric antigen receptor (CAR) T cell therapy is a form of cellular immunotherapy for the treatment of hematological malignancies, in which a patient’s T lymphocytes are genetically modified ex vivo to express a CAR that enables recognition and elimination of tumor cells expressing a specific target antigen. The CAR molecule is composed of four key components: an extracellular antigen-binding domain, a hinge domain, a transmembrane (TM) domain, and at least one intracellular signaling domain. While previous research has focused on the role of co-stimulatory domains in CAR-T cell activation, in this master’s thesis we investigated the impact of hinge and TM domains on CAR-T cell functionality, given their known involvement in CAR surface expression, intracellular signaling and cytokine secretion. Using molecular cloning techniques, we generated CAR constructs in E. coli containing various combinations of native and alternative hinge and TM domains. These constructs were introduced into Jurkat E6.1 cell line via electroporation or lentiviral transduction. Expression was assessed using flow cytometry and confocal microscopy, while functionality was assessed in co-culture with Raji tumor cells using enzyme-linked immunosorbent assay (ELISA). Our results demonstrated that hinge and TM domains influence both surface expression of the CAR and the secretion level of human interleukin 2 (hIL-2) upon stimulation with tumor cells. Additionally, we transduced Jurkat TPR cells, which stably express three reporter proteins, enabling simultaneous monitoring of transcription factor activity critical for CAR-T cell activation (NF-κB, NFAT, and AP-1). Stimulation of these cells with tumor cells revealed activation of all three signaling pathways, with certain combinations of alternative hinge and TM domains enhancing transcriptional activity compared to commercially used domains. These findings indicate that the choice of hinge and TM domains significantly affects CAR-T cell performance and may represent a strategy to optimize therapeutic efficacy and safety. However, further evaluation of the clinical potential of individual domains and constructs in primary T cells is required.
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