Browsing by Author "Kozlova, Anastasia"

Chimeric antigen receptor (CAR) T cells are genetically modified usually autologous T cells expressing de novo designed CAR that binds a specific antigen on the surface of the cancer cells, inducing T cell receptor-independent activation and cytotoxic response against the targeted cancer cells. While CAR T cells have been shown to offer effective treatment in acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and multiple myeloma, several resistance mechanisms can lead to CAR T cell exhaustion characterized by impaired functions and the expression of inhibitory receptors. The Finnish Red Cross Blood Service has developed novel CARs, differing in structure from the ones currently published. Since the evasion of CAR T cell exhaustion is considered one of the key objectives in the development of CAR T cell therapy, this Master’s thesis project aimed to create a working method to determine the exhaustion of CAR T cells in vitro after long-term repeated stimulation. In order to induce and measure exhaustion, CAR T cells were produced and activated ex vivo in the presence of IL-2 or IL-7/IL-15 cytokines, cultured long-term and repeatedly stimulated by exposure to target cells. CAR T cell cytotoxicity and expansion were determined and the expression of various inhibitory receptors was analyzed. The method enabled the comparison of the designed CAR T cell candidates and the positive control CD19-CD28ζ CAR T cells in long-term cytotoxic potency. In addition, it helped to reveal the surprising difference between IL-2 and IL-7/IL-15 cytokines and their impact on CAR T cell exhaustion. Although CAR T cells produced with IL-2 had poorer expansion during CAR T cell production than CAR T cells produced with IL-7/IL-15, they showed lower expression of exhaustion-related markers supported by better survival, proliferation and cytotoxic activity during long-term repeated stimulation assay.