Figure 2.
Production strategies and cellular sources for CAR-NK,CAR-M and CAR-γδ T therapeutics. The NK92 cell line represents a widely utilized source for CAR-NK manufacturing due to its capacity for indefinite in vitro expansion and resilience to freeze-thaw cycles. However, as a malignant line derived from natural killer cell lymphoma, CAR-engineered NK92 cells require γ-irradiation prior to clinical infusion to eliminate proliferative risks. Primary NK cells, by contrast, are isolated from PBMCs of healthy donors or UCB via immunomagnetic selection, followed by activation, genetic modification with viral vectors (e.g., lentiviral or retroviral systems), and expansion in cytokine-enriched media to achieve clinical-grade quantities. Alternatively, CD34+ HPCs can be differentiated into NK lineages using defined cytokine cocktails, subsequently engineered with CARs, and amplified in vitro for therapeutic use. Recent advances highlight induced pluripotent stem cells (iPSCs) as a scalable platform for “off-the-shelf” CAR-NK production. iPSCs undergo sequential differentiation into CD34+ HPCs and functional NK cells, with CAR integration achievable at the pluripotent stage. This approach enables standardized generation of CAR-iPSC-derived NK cells, circumventing donor variability. For CAR-Ms, primary macrophages are typically isolated from donor PBMCs or differentiated in vitro from THP-1 cell lines, fibroblast-derived progenitors, or iPSCs. Following isolation or differentiation, these cells are transfected with CAR-encoding constructs and functionally validated prior to adoptive transfer. iPSC-derived CAR-Ms further offer potential for renewable, genetically uniform therapeutic batches. For CAR-γδ T, primary γδ T are typically isolated from donor PBMCs. Created using BioRender.com.