Generation and functional characterization of CAR exosomes

Chimeric antigen receptor (CAR) T cells have attracted substantial attention in recent years as an emerging therapy for hematological and non-hematological malignancies. Despite the rapid and robust clinical responses, unexpected toxicity, such as cytokine release syndrome, still remains a major concern in this therapy. Moreover, the intrinsic ability of tumors to evade immune responses could lead to treatment failure especially in patients with solid tumors. These obstacles together highlight a need to improve current CAR-T therapy. Exosomes are small extracellular vesicles secreted by almost all cell types and have the capability of trafficking cargos to mediate many physiological/pathophysiological processes. Therefore, researchers have been trying to utilize exosomes as highly effective carriers to deliver various therapeutic agents to target cells. We reported that CAR-T cells release extracellular vesicles with the stimulation of antigens, mostly in the form of exosomes that carry CARs on their surface. These CAR exosomes express a high level of cytotoxic molecules and therefore inhibit tumor growth in an antigen-specific manner. Besides, CAR exosomes do not express programmed cell death protein 1 (PD1), and thus could circumvent the immunosuppressive mechanism caused by tumor cells. More importantly, the administration of CAR exosomes exhibited lower risk compared with CAR-T therapy in a preclinical in vivo model of cytokine release syndrome. All these advantages of CAR exosomes suggest that they may be promising therapeutic agents against tumors. Here, we describe the methods to generate CAR exosomes and the functional characterization of these therapeutic nano-vesicles.