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

## TL;DR

Engineered CAR-NKT extracellular vesicles effectively target and suppress tumors while reducing toxicity, offering a promising new approach to cancer immunotherapy.

## Contribution

A novel nanobody-based CAR-NKT extracellular vesicle therapy is introduced as a safer and more effective alternative to traditional CAR-NKT cell therapy.

## Key findings

- CARTM4SF1-EVs selectively target TM4SF1-expressing tumor cells and suppress tumor growth and metastasis in HCC models.
- CARTM4SF1-EVs induce immunogenic cell death and enhance CD8⁺ T cell activity within the tumor microenvironment.
- CARTM4SF1-EVs synergize with immune checkpoint blockade to elicit durable antitumor immune memory.

## 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 CARTM4SF1‐NKT cells. Moreover, CARTM4SF1‐extracellular vesicles (EVs) therapy as an optimized alternative to direct CAR‐NKT cell administration is introduced. Compared with conventional CARTM4SF1 engineered cells, CARTM4SF1‐EVs demonstrated superior antitumor efficacy while significantly reducing toxicity. This findings revealed that CARTM4SF1‐EVs selectively targeted TM4SF1‐expressing tumor cells in both in vitro and in vivo models. In hepatocellular carcinoma (HCC) mouse models, CARTM4SF1‐EVs induced immunogenic cell death (ICD) and effectively suppressed tumor growth and metastasis. The therapeutic efficacy of CARTM4SF1‐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, CARTM4SF1‐EVs synergized with Immune Checkpoint Blockade (ICB) therapy, leading to durable antitumor immune memory. Collectively, these findings establish CARTM4SF1‐EVs therapy as a safe and effective strategy for targeted cancer immunotherapy, underscoring its potential for clinical application.

TM4SF1‐nanobody engineered CAR‐natural killer T–derived extracellular vesicles (CARTM4SF1‐EVs) provide a cell‐free alternative to CAR‐NKT therapy, achieving potent, targeted antitumor activity with reduced toxicity. CARTM4SF1‐EVs induce immunogenic cell death, remodel the tumor microenvironment, and enhance CD8⁺ T‐cell antitumor immunity. They further synergize with immune checkpoint blockade, providing a safe and effective strategy for cancer immunotherapy.