Distinct effector functions and synergy of CAR mRNA-engineered T cells and macrophages in the clearance of CD19 leukemia cells.

[INTRODUCTION] Chimeric antigen receptor (CAR) technology represents a groundbreaking advancement in gene therapy. While CAR-T cells are approved for specific types of leukemia, the therapeutic potential of CAR-macrophages remains largely unexplored. Additionally, potentially synergistic effects between CAR-T cells and CAR-macrophages in cancer cell clearance are still elusive.

[OBJECTIVES] This study aimed to investigate the efficiency and mechanisms of human CAR-T cells and CAR-macrophages for clearing cancer cells using co-culture experiments.

[METHODS] We encapsulated a second generation CD19 CAR mRNA or enhanced green fluorescent protein (Egfp) encoding control mRNA into lipid nanoparticles (LNP) using different methods. We transfected human primary T cells or macrophages with these LNP and studied their CAR expression using flow cytometry or confocal microscopy. Killing and phagocytosis assays were conducted to evaluate the impact of CAR-T cells and CAR-macrophages on CD19 NALM6 leukemia and CD19 control cells. Motility analyses of macrophages, T cells, and cancer cells were done using live-cell imaging.

[RESULTS] Activated T cells upregulated the low-density lipoprotein receptor (LDLR), resulting in higher CAR surface expression post-transfection than in non-activated T cells. CAR-T cells efficiently induced death of lymphoma cells. CAR-macrophages cleared cancer cells mainly via phagocytosis and CAR mRNA altered the expression of both M1 and M2-associated genes. Interestingly, CAR macrophages also upregulated the Signal regulatory protein alpha (SIRPA), a receptor of the CD47 ligand expressed by cancer cells, and knockdown of SIRPA by siRNA efficiently counteracted this phenomenon. We noted a decrease in the motility of macrophages, T cells, and cancer cells in co-cultures containing CAR-transfected effector cells. Co-culturing CAR-T cells with CAR-macrophages synergistically enhanced tumor cell eradication.

[CONCLUSION] Our findings underscore the superior cancer cell-killing capability of CAR-T cells. Additionally, CAR-macrophages play a crucial role in clearing cancer cells through phagocytosis, thereby synergistically supporting the activity of CAR-T cells.