Background <p>Autologous chimeric antigen receptor (CAR) expressing T-Cells (CAR-T) have been efficiently used in hematological malignancies but their efficacy in solid tumors remains limited. CAR therapies via the use of macrophages, offer a promising avenue due to their unique ability to infiltrate tumors and to initiate phagocytosis.</p> Methods <p>To generate a preclinical model of CAR-Macs, we have designed a novel CAR-construct to target EGFR^VIII-expressing glioblastoma cells (DK-MG) using THP-1 monocytic cell line able to differentiate towards macrophages. The CAR structure comprises a ScFv recognizing specifically EGFR^VIII and MEGF10 intracellular domain which enhances tethering and phagocytosis activity.</p> Results <p>THP-1 cell line expressing CAR and control constructs 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 macrophages or CAR-Macs in the presence of target DK-MG cells. Confocal microscopy experiments revealed highly efficient phagocytosis of DK-MG EGFR^VIII cells by CAR-Macs (~ 60%) as compared to WT cells (~ 15%). The killing potential of the anti-EGFR^VIII CAR-Macs against the glioblastoma cell line has also been observed using video microscopy. ELISA and LDH assays performed in co-culture supernatants, showed an increase of IL-6 and LDH levels suggesting efficient cytotoxicity via CAR-Macs. Transcriptome analyses performed in purified CAR-Macs, revealed evidence of a molecular signature in favor of successful phagocytosis.</p> Conclusions <p>The model described in this work is suitable for allowing translation to iPSC-derived macrophages to generate clinically applicable future cell therapy approaches in solid tumors expressing mutated variant EGFR^VIII.</p>