Investigation of the immunotherapeutic effect of cord blood-derived NK cells on CD-47 knock-out non-small cell lung cancer cells by CRISPR-Cas9 technique

One of the escape routes from the immune surveillance system is to prevent the recognition of cancer cells by the immune system by reducing NK (Natural Killer) cell activation. Specifically, one of the signals that is important in this escape is the SIRP-α (Signal Regulator protein-α/) /CD47 (Cluster of Differentiation-47) interaction. By increasing CD47 expression, cancer cells show a synaptic interaction with SIRPα in immune system cells and evade immune surveillance. Based on this, in this study, the aim was to increase the recognition of cancer cells by NK cells by knocking out CD47 on non-small cell lung cancer cells (A549) using the CRISPR/Cas9 technique, thereby blocking the SIRPα/CD47 axis and making the cancer cells an open target. To achieve this goal, in this thesis, NK cells were first isolated and cultured from cord blood. Subsequently, CD47, which is an immune escape ligand on tumor cells and expressed on non-small cell lung cancer cells (A549), was knocked out using the CRISPR/Cas9 technique. The aim is to block the interaction of CD47 gene knock-out non-small cell lung cancer cells (A549) with SIRPα and to block the "don't eat me" signaling. Based on the findings obtained, a significant difference (p<0.0001) was observed in terms of cell viability between the groups as a result of co-culture of increased effector NK cell ratios with target A549 cells for 24 and 48 hours. In addition, as a result of CD47 silencing, NK cells exhibited a significant cytotoxic effect against non-small cell lung cancer cells (A549) in a dose-dependent manner. The study found that blocking the SIRPα/CD47 axis enhances the cytotoxic effect of cord blood-derived NK cells on genetically modified (CD47-/-) non-small cell lung cancer cells, suggesting that cord blood-derived NK cells could serve as an immunotherapeutic tool.