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.