Cell-free CAR therapy: BCMA-CAR-T cell-derived exosomes exhibit potent anti-myeloma activity Free

Introduction: Chimeric antigen receptor (CAR) T-cell therapy targeting BCMA has shown remarkable clinical efficacy in patients with multiple myeloma (MM). However, the manufacturing process for CAR-T cells remains technically complex, costly, and time-consuming. These limitations make large-scale production, storage, and distribution difficult, thereby restricting broader access for patients. Recent publications demonstrate that exosome-based therapies have emerged as a promising cell-free approach in cancer treatment, owing to their ability to mimic the functional properties of their cells of origin while offering improved safety, stability, and ease of storage and delivery. Particularly, exosomes derived from CAR-T cells retain components of the CAR machinery and tumor-targeting capacity. These CAR-containing exosomes can mediate direct cytotoxic effects against tumor cells, induce immune activation, and circumvent several limitations. On this backdrop, we investigated BCMA-CAR-T cell-derived exosomes as a novel cell-free immunotherapy for MM.

Methods: A second-generation BCMA-CAR construct was designed, incorporating a BCMA-scFv, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. Human T cells were transduced with lentivirus encoding the CAR construct. BCMA CAR-T cells were stimulated with MM cells at an effector-to-target ratio of 5:1 for five days. Exosomes were isolated from the culture supernatant through serial differential centrifugation: 300×g for 10 min, 2000×g for 10 min, and 10,000×g for 30 min to remove cells and debris. The supernatant was further ultracentrifuged at 100,000×g for 90 min, and the resulting pellet was resuspended in 10 mM HEPES. To eliminate contaminating proteins, an additional ultracentrifugation step at the same speed was performed. Exosome morphology and size were assessed using a JEM-1400Flash transmission electron microscope with uranyl acetate negative staining. BCMA-CAR expression in exosomes was verified by Western blotting. For cytokine detection, exosomes were lysed with 0.3% Triton X-100. IFN-γ, TNF-α, and IL-2 levels were quantified using ELISA. To assess cytotoxicity, OPM2, MM.1S, H929, and OPM2^BCMAKO cells were co-cultured with 50, 100, 200, 300, and 500 µg/mL CAR exosomes at 37 °C for 24 hours. Cytolytic activity was evaluated using an LDH release assay.

Results: BCMA-CAR-T cells (1×10⁷/mL) were stimulated in 50 mL of medium containing OPM2 cells (5×10⁶/mL), yielding 1,800µg of exosomes suspended in 1 mL of HEPES. The nano-sized exosomes isolated from the culture supernatant were typically cup-shaped, with an average diameter of 109 ± 9 nm. Western blotting confirmed the presence of BCMA-CAR on the exosome membrane. CAR exosomes induced dose-dependent lysis of OPM2 cells, reaching 38.0% at 500µg/mL, while showing minimal cytotoxicity toward OPM2^BCMAKO cells (2.8%). At the same concentration, BCMA-CAR exosomes demonstrated significantly greater cytotoxicity than control T cell-derived exosomes (38.0% vs 1.8%). Cytolytic activity comparable to OPM2 was also seen in MM.1S (54.8% vs 3.6%) and H929 (30.4% vs 4.8%). No significant difference in cytotoxicity was observed between CAR and control exosomes when co-cultured with OPM2^BCMAKO cells. CAR exosomes (100 µg) pretreated with Triton X-100 released measurable amounts of cytokines, including IFN-γ (228.0 pg), TNF-α (424.7 pg), and IL-2 (262.5 pg). Moreover, co-culture of CAR exosomes (500 µg/mL) with OPM2 cells significantly increased cytokine release compared to control exosomes: IFN-γ (503.3 vs 65.0 pg/mL), TNF-α (765.0 vs 27.9 pg/mL), and IL-2 (500.1 vs 54.3 pg/mL). This cytokine elevation was not observed when co-cultured with OPM2^BCMAKO cells.Conclusion: In this study, BCMA-targeted CAR exosomes obtained from CAR-T cells demonstrate strong cytotoxic activity to BCMA-expressing MM in vitro. Compared to traditional CAR-T cell therapy, CAR exosomes offer several advantages. They are cell-free, making them easier to manufacture, store, and deliver. Moreover, our CAR exosomes outperformed control exosomes, showing better cytotoxic and immunostimulatory capabilities, thus highlighting the added value of CAR engineering in exosome-based therapy. We are currently conducting ongoing in vivo studies to assess further the anti-MM efficacy and safety profile of CAR exosomes. In summary, our findings support the potential of CAR exosomes in a cell-free immunotherapeutic strategy for MM.