CAR-NK cell therapy for overcoming antigen heterogeneity and immunosuppressive tumor microenvironments

• CAR-NK enable MHC-independent, off-the-shelf immunotherapy. • Armored constructs enhance persistence and cytokine secretion. • CRISPR-edited CISH knockout improves antitumor activity. • Non-viral delivery platforms increase safety and scalability. • Clinical trials show efficacy with manageable toxicity. Chimeric antigen receptor engineered natural killer cells (CAR-NK) have emerged as a transformative strategy in cancer immunotherapy, offering MHC-independent cytotoxicity, reduced graft-versus-host disease risks, and allogeneic “off-the-shelf” applicability. This review synthesizes advancements in CAR-NK design, including fourth-generation “armored” constructs with cytokine secretion, metabolic reprogramming via mTOR/c-Myc modulation, and CRISPR-mediated knockout of inhibitory checkpoints (CISH). Clinical trials demonstrate efficacy in hematologic and solid tumors, with HER2-, B7-H3-, and NKG2D-targeted CAR-NK cells showing manageable toxicity and minimal cytokine release syndrome. Innovations such as non-viral gene delivery (Sleeping Beauty transposon, mRNA electroporation) and combinatorial approaches (immune checkpoint inhibitors, chemokine receptor integration) enhance tumor infiltration and persistence. Despite progress, challenges persist, including tumor heterogeneity, immunosuppressive tumor microenvironment barriers, and scalability of manufacturing protocols. This review critically evaluates preclinical and clinical data, highlighting emerging targets (EpCAM, CEA), engineering paradigms (logic-gated CARs, trogocytosis-mediated transfer), and strategies to counteract antigen loss and metabolic stress. By addressing knowledge gaps in TME modulation and standardizing clinical protocols, this work aims to catalyze interdisciplinary efforts to advance safer, durable CAR-NK therapies for refractory cancers.