Development of a novel Chimeric Antigen Receptor (CAR) for its use in Natural Killer (NK) cells for cancer treatment and beyond

The use of CAR technologies has revolutionized cancer treatment. Their unprecedented efficacy against B cell malignancies has opened the doors for a lot of excitement and research in the field. These synthetic receptors are composed of an antigen recognition domain and an activating signaling domain that allows immune cells to direct their function in an antigen dependent manner.Despite all the potential of CAR T cell technologies, their efficacy is accompanied by severe, even life-threatening toxicities. Moreover, there is still a considerable number of patients that do not respond to the therapy. On top of this, their large scale applicability is hindered by their production, as autologous T cell manufacturing is a complex, lengthy and costly process. Moreover, cancer patients often present deficient or exhausted immune cells, which can lead to failures to manufacture, and therefore they cannot be used in a therapeutic perspective.Much research has focused on the prevention and treatment of the CAR-associated toxicities and on the further improvement of this technology. An interesting approach is the use of other immune cell types, such as Natural Killer (NK) cells. NK cells are a promising platform for CAR therapies due to their excellent safety profile, their natural role to kill tumor cells, and the possibility to be administered in an allogenic manner. Despite the increasing interest in using NK cells for CAR therapies, the constructs used in this setting are still highly based on the ones developed for T cells. With the intention to develop a new CAR construct with improved function for NK cells, the presented thesis looked into their endogenous signaling, and how 2B4, an important NK cell receptor, interacts with LAT for its immune activating function. LAT serves as a signaling hub between proximal and distal TCR signaling, which plays a major role in immune cell activation. This thesis presents the development and optimization of a CAR construct based on LAT signaling and observations of improved in vitro repetitive tumor killing. This was accompanied by better tumor control in vivo.The efficacy of this construct in T cells was then tested. Unaltered, this construct induced poorer tumor control than commonly used CD28-CD3ζ CAR constructs. However, after tuning the activity of the receptor with the inclusion of a co-stimulatory domain and a mutation that induces faster phosphorylation, similar activity against cancer cells in vitro and in vivo in T cells could be achieved.Looking at the potential novel applications of CAR technologies beyond cancer, it was demonstrated that the LAT-based CAR could also induce regulatory function when used in regulatory T cells, potentially allowing for its use to treat autoimmune diseases. It could also be shown that the LAT-based CAR can induce phagocytosis when expressed in a monocytic cell line. Finally, the great potential that the LAT-based CAR offers to be used in NK cells in order to treat cardiac fibrosis could also be demonstrated.