DP028 | CD19.CAR-T CELL-DERIVED EXTRACELLULAR VESICLES RETAIN CAR EXPRESSION AND EXHIBIT CYTOTOXIC ACTIVITY AGAINST LEUKEMIC CELLS, SUPPORTING THEIR ROLE IN ANTITUMOR THERAPY.

CAR-T cell therapy has revolutionized the treatment of hematologic malignancies. Nevertheless, significant challenges remain, particularly concerning its severe adverse effects. Extracellular vesicles (EVs) emerged as promising therapeutic agents due to their better safety profiles. Given that CAR-T cell-derived EVs may replicate the anti-tumor activity of CAR-T cells, this study aimed to characterize the phenotype and functions of CD19.CAR-T cell-derived EVs and assess their therapeutic potential. A cohort of 22 patients infused with CD19.CAR-T cells was monitored for the presence of circulating CD19.CAR+-T cell-derived EVs (CD19.CAR+EVs). EVs were isolated from either pre-infusion CD19.CAR-T cell products (n = 3) or from circulating CD19.CAR-T cells (n = 3) collected from infused patients. EVs were purified and co-cultured with two CD19+ cell lines (Raji and SUP-B15). Anti-tumor activity of CD19.CAR-T and B7H3.CAR-T cell-derived EVs was assessed using MTT assays and cytotoxicity assays. Morphological characterization of CD19.CAR-T EVs was carried out via nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and atomic force microscopy (AFM). Phenotypic analysis included flow cytometry and proteomic profiling. CD19.CAR+ EVs were detectable in peripheral blood for up to two years post-infusion, indicating the long-term persistence of their parent CAR-T cells. Notably, an early decline in circulating CD19.CAR+ EV levels was associated with CAR-T therapy failure. The characterization of CD19.CAR-T EVs demonstrated a spherical morphology with an average size of 133.1 ± 65.5 nm. These EVs expressed canonical EV markers (CD63 and Flotillin-1) and lacked typical contaminants (i.e. cytochrome C and apolipoproteins). Notably, CD19.CAR-T EVs exhibited higher levels of CAR than their parental cells and contained a pro-apoptotic protein cargo potentially responsible for their cytotoxic activity. CD19.CAR-T EVs significantly reduced the viability of CD19+ target cells in a dose-dependent manner. At a dose of 0.015 μg of EV protein per target cell, circulating CD19.CAR-T EVs achieved killing rates of 20.3% (± 3.8) for Raji cells and 37.8% (± 7.0) for SUP-B15 cells. Pre-infusion CD19.CAR-T EVs induced killing rates of 42.0% (± 12.3) and 35.8% (± 13.8) for Raji and SUP-B15, respectively. These findings suggest that CD19.CAR-T cell-derived EVs could serve as a novel CAR-T cell-based therapeutic product. By directly targeting and killing leukemic cells, they offer a potentially safer alternative that may be administered independently of CAR-T cells.