Engineered CAR‐NKT Extracellular Vesicles Suppress Tumor Progression and Enhance Antitumor Immunity

Chimeric antigen receptor-engineered natural killer T (CAR-NKT) cell therapy represents a promising and innovative strategy in cancer immunotherapy, but is limited by acute toxicity and adverse effects, restricting broader clinical application despite durable responses. In this study, a novel nanobody targeting TM4SF1 is developed, which replaced the conventional single-chain variable fragment (scFv) in the design of CAR^TM4SF1-NKT cells. Moreover, CAR^TM4SF1-extracellular vesicles (EVs) therapy as an optimized alternative to direct CAR-NKT cell administration is introduced. Compared with conventional CAR^TM4SF1 engineered cells, CAR^TM4SF1-EVs demonstrated superior antitumor efficacy while significantly reducing toxicity. This findings revealed that CAR^TM4SF1-EVs selectively targeted TM4SF1-expressing tumor cells in both in vitro and in vivo models. In hepatocellular carcinoma (HCC) mouse models, CAR^TM4SF1-EVs induced immunogenic cell death (ICD) and effectively suppressed tumor growth and metastasis. The therapeutic efficacy of CAR^TM4SF1-EVs is primarily attributed to their ability to remodel the immunosuppressive tumor microenvironment (TME), notably by enhancing CD8⁺ T cell activity and eliciting robust antitumor immune responses. Furthermore, CAR^TM4SF1-EVs synergized with Immune Checkpoint Blockade (ICB) therapy, leading to durable antitumor immune memory. Collectively, these findings establish CAR^TM4SF1-EVs therapy as a safe and effective strategy for targeted cancer immunotherapy, underscoring its potential for clinical application.