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

Abstract 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.