Cadinanos-Garai Amaia, Flugel Christian L, Cheung Anson, Jiang Enzi, Vaissié Alix, Abou-El-Enein Mohamed
USC/CHLA Cell Therapy Program, University of Southern California and Children's Hospital of Los Angeles, Los Angeles, CA 90033, USA.
USC/CHLA Cell Therapy Program, University of Southern California and Children's Hospital of Los Angeles, Los Angeles, CA 90033, USA; Berlin Center for Advanced Therapies, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 13353 Berlin, Germany.
Mol Ther. 2025 May 7;33(5):2291-2309. doi: 10.1016/j.ymthe.2025.04.006. Epub 2025 May 1.
Despite the notable success of chimeric antigen receptor (CAR) T cell therapies in hematological malignancies, clinical outcomes remain variable, making it critical to understand how manufacturing influences product composition and function. We developed a 36-marker spectral flow cytometry panel enabling integrated profiling of phenotypic, metabolic, and functional attributes across CAR T cell production. Mid-expansion products (day 5) retained stem-like, metabolically active CD4 Th1 subsets with high proliferative capacity, whereas prolonged culture (day 10) enriched terminally differentiated CD8 Tc1 cells and NK-like T cell populations. CAR and CAR T cells showed similar differentiation trajectories, suggesting that culture conditions may have a larger impact on phenotypic remodeling than CAR integration. Upon antigen encounter and restimulation, day 5 and day 10 products showed comparable cytotoxicity, while differing in their activation and checkpoint profiles. Cryopreservation modestly affected stem cell memory, activation, and metabolic markers but preserved overall phenotype and cytotoxic function. These findings establish a high-dimensional framework for mapping CAR T cell dynamics to support manufacturing optimization and next-generation cell therapy design.
尽管嵌合抗原受体（CAR）T细胞疗法在血液系统恶性肿瘤治疗中取得了显著成功，但临床结果仍存在差异，因此了解生产过程如何影响产品组成和功能至关重要。我们开发了一个包含36个标记物的光谱流式细胞术检测板，能够对CAR T细胞生产过程中的表型、代谢和功能属性进行综合分析。中期扩增产物（第5天）保留了具有高增殖能力的干细胞样、代谢活跃的CD4 Th1亚群，而延长培养时间（第10天）则富集了终末分化的CD8 Tc1细胞和NK样T细胞群体。CAR和CAR T细胞表现出相似的分化轨迹，这表明培养条件对表型重塑的影响可能比CAR整合更大。在遇到抗原并再次刺激后，第5天和第10天的产物表现出相当的细胞毒性，但其激活和检查点特征有所不同。冷冻保存对干细胞记忆、激活和代谢标记物有轻微影响，但保留了整体表型和细胞毒性功能。这些发现建立了一个高维框架，用于描绘CAR T细胞动态，以支持生产优化和下一代细胞疗法设计。
