Lanuti Paola, Guardalupi Francesco, Corradi Giulia, Florio Rosalba, Brocco Davide, Veschi Serena, Pennese Elsa, De Bellis Domenico, D'Ascanio Francesca, Piro Anna, De Lellis Laura, Simeone Pasquale, Cufaro Maria Concetta, Pilato Serena, D'Amario Isabella, Villanova Ida, Di Francesco Barbara, Di Re Lucia, Verginelli Fabio, Pieragostino Damiana, Salutari Pressede, Colasante Fabrizia, Natale Annalisa, Mattoli Maria Vittoria, Vespa Simone, Fontana Antonella, Giancola Raffaella, Fabi Bianca, Baldoni Stefano, Santarone Stella, Restuccia Francesco, Tinari Nicola, Del Boccio Piero, Cama Alessandro, Di Ianni Mauro
University "G. d'Annunzio", Chieti-Pescara, Chieti, Italy.
University of Chieti-Pescara, Pescara, Italy.
Blood Adv. 2025 Feb 4. doi: 10.1182/bloodadvances.2024014860.
Chimeric Antigen Receptor (CAR) T cell-derived Extracellular Vesicles (EVs) might represent a new therapeutic tool for boosting CAR-T cell antileukemic effects. Here, 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), which were then separated and functionally characterized for their killing abilities. A GMP-compliant separation method was also developed. Results demonstrated that CD19.CAR+EVs were detectable in peripheral blood up to 2 years after infusion indicating long-lasting persistence of their parental cells. Notably, early decreases of circulating CD19.CAR+EVs concentrations correlated with failure of CAR-T therapy. Circulating CD19.CAR+EVs displayed a median size (SD) of 133.1±65.5 nm and carried a pro-apoptotic protein cargo. These EVs expressed higher CAR levels than their parental cells. Furthermore, CD19.CAR+EVs did not activate heterologous T cells and produced significant, specific and dose-dependent cytotoxic effects on CD19+ cell lines and on primary cells. The new GMP-compliant EV isolation method allowed a recovery of 63±5.7 % of CD19.CAR+EVs. A deeper analysis of the different protein cargoes carried by EVs derived from different CAR-T cell subpopulations identified a pro-apoptotic functional pathway linked to CD8+LAG-3+ EVs. Overall, our data indicate that CD19.CAR+EVs may be proposed as promising dynamic new biomarkers of CAR-T cell activity and, contributing to the direct killing of the leukemic target, represent a new product with a strong therapeutic potential that could be infused independently from CAR-T cells.
嵌合抗原受体（CAR）T细胞衍生的细胞外囊泡（EVs）可能代表一种增强CAR-T细胞抗白血病作用的新治疗工具。在此，对22名输注CD19.CAR-T细胞的患者队列进行监测，以检测循环中CD19.CAR+T细胞衍生的EVs（CD19.CAR+EVs）的存在，然后分离这些EVs并对其杀伤能力进行功能表征。还开发了一种符合药品生产质量管理规范（GMP）的分离方法。结果表明，输注后长达2年外周血中均可检测到CD19.CAR+EVs，表明其亲代细胞具有持久的持久性。值得注意的是，循环中CD19.CAR+EVs浓度的早期下降与CAR-T治疗失败相关。循环中的CD19.CAR+EVs中位大小（标准差）为133.1±65.5 nm，并携带促凋亡蛋白货物。这些EVs表达的CAR水平高于其亲代细胞。此外，CD19.CAR+EVs未激活异源T细胞，并对CD19+细胞系和原代细胞产生显著、特异性和剂量依赖性的细胞毒性作用。新的符合GMP的EV分离方法可回收63±5.7%的CD19.CAR+EVs。对来自不同CAR-T细胞亚群衍生的EVs所携带的不同蛋白质货物进行更深入分析后发现，一条与CD8+LAG-3+ EVs相关的促凋亡功能途径。总体而言，我们的数据表明，CD19.CAR+EVs可被视为CAR-T细胞活性有前景的动态新生物标志物，并且由于其有助于直接杀伤白血病靶点，代表一种具有强大治疗潜力的新产品，可独立于CAR-T细胞进行输注。
