A Novel Chimeric Antigen Receptor (CAR) - Strategy to Target EGFR^VIII-Mutated Glioblastoma Cells via Macrophages

Autologous chimeric antigen receptor (CAR) expressing T-Cells (CAR-T) have been efficiently used in hematological malignancies but their efficacy in solid tumors remain limited. CAR therapies via the use of macrophages, offer a promising avenue due to their unique ability to infiltrate tumors and to initiate phagocytosis. To generate a model of CAR-macrophage for cancer therapy, we have designed a novel CAR monocyte-construct to target EGFRVIII-expressing glioblastoma cells (DK-MG) using THP-1 monocytic cell line able to differentiate towards macrophages. The CAR structure comprises a ScFv recognizing specifically EGFRVIII and MEGF10 intracellular domain which enhances tethering and phagocytosis activity. THP-1 cell line expressing CAR and control construct were generated via lentiviral transduction followed by generation of monocytes and CAR-expressing M1 macrophages (CAR-MACs). To evaluate their ability of phagocytosis, we co-cultured THP-1 derived WT or CAR-MACs with the target DK-MG cells. Confocal microscopy experiments revealed highly efficient phagocytosis of DK-MG EGFRVIII cells by CAR-MACs (~60%) as compared to WT cells (~15%). The killing potential of the anti-EGFRVIII CAR-Mac against the glioblastoma cell line DKMG has also been demonstrated using video microscopy. ELISA assays performed with co-culture supernatants, showed a significant increase of IL-6 and LDH in the presence of CAR-mediated cell killing. Transcriptome analyses performed after co-culture of FACS-sorted macrophages, revealed evidence of a signature in favor of successful phagocytosis. Thus, this model is suitable for translation to iPSC-derived macrophages to generate a clinically applicable future cell therapy approaches in all solid tumors expressing mutated EGFRVIII.