P08.15.A DEVELOPMENT OF CAR MACROPHAGES AND MICROGLIA AS A NOVEL TREATMENT AGAINST GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. It is characterized by a poor prognosis and guaranteed relapse despite treatment. Immunotherapies like checkpoint inhibitors and chimeric antigen receptor (CAR) T cells have improved prognosis in many other cancers but have failed in GBM. Our project aims to engineer CAR macrophages (CAR M) and CAR microglia (CAR MG) as a new treatment approach for GBM patients. MATERIAL AND METHODS Macrophages are inherently resistant to lentiviral transduction. To increase the transduction rate, we created a modified lentivirus expressing the accessory protein Vpx. We then transduced primary CD14+ monocytes from healthy donors and differentiated them into CAR M, armed with a CAR targeting the GBM-specific antigen EGFRvIII. Additionally, we started a production pipeline of induced pluripotent stem cell (iPSC)-derived microglia. Embryoid bodies were formed using mTesR+ medium supplemented with BMP-4, SCF and VEGF-121. We generated primitive macrophage precursors (PMPs) by adding M-CSF and IL-3. Final microglia differentiation was achieved with IL-34 and GM-CSF. RESULTS Using our modified lentiviral construct, we achieved a transduction efficacy of primary macrophages of 57.6%; a 50-fold increase compared to baseline transduction with an unmodified lentivirus. After multiple rounds of testing a range of different conditions, we were able to develop and consolidate a protocol that yields both reliable transduction rates and satisfactory macrophage viability. Furthermore, we generated iPSC-derived cells that display a microglia-like morphology in vitro. Embryoid bodies reliably gave rise to large numbers of PMPs. After final differentiation, the differentiated microglia were adherent and depicted their characteristic ramified phenotype. We successfully lentivirally transduced a first batch of iPSCs and are differentiating these to CAR microglia. CONCLUSION We developed a protocol achieving reliable genetic modification of primary macrophages and established the production of iPSC-derived microglia in our laboratory. Combined, these skills provide us with a foundation to further develop and refine our generation of CAR M and CAR MG, a potential new immunotherapy for GBM. In a next step, we will carry out functional tests on CAR M and CAR MG to assess their anti-tumor effects in vitro and in vivo.