Sierro-Martínez Belén, Guijarro-Albaladejo Beatriz, Fernández-Cisnal Ricardo, Rodríguez-Gil Alfonso, Hernández-Díaz Paola, de la Rosa-Garrido María, Carrasco-Brocal Inmaculada, Lara-Chica Maribel, Escamilla-Gómez Virginia, Muñoz-García Raquel, Bella Ángela, García-Cózar Francisco Jose, Pérez-Simón Jose Antonio, García-Guerrero Estefanía
Instituto de Biomedicina de Sevilla, IBiS/Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain.
Instituto de Investigación E Innovación Biomédica (INiBICA), Cádiz, Spain.
J Transl Med. 2025 Aug 19;23(1):942. doi: 10.1186/s12967-025-06899-0.
Despite the promising results of CAR-T cell therapy, still a significant proportion of patients experience treatment failure due to absence of response, progression or treatment-related toxicities. Our research aims to investigate how the CAR-T cell product characteristics and particularly the percentage of CAR-negative cells present in the infusion product affects its efficacy and safety.
CAR-positive (CAR-pos) and CAR-negative (CAR-neg) cells were analyzed during in vitro expansion with IL-2 or IL-7/IL-15. FACS-sorted populations were assessed for cytotoxic effect and secretion of cytokines. Transcriptomic analyses were conducted for CAR-pos and CAR-neg populations.
A significant reduction in the percentage of transduced (CAR-pos) T cells was observed throughout the in vitro cell culture, where CAR-neg T cells displayed greater expansion along the procedure compared to CAR-pos T cells, using either IL-2 or IL-7/IL-15. CAR-pos T cells exhibited increased expression of activation and exhaustion markers. Comparative analyses of CAR-pos and CAR-neg showed an increased cytotoxic effect in purified samples with 100% CAR-pos T cells compared to non-purified product. Quantification of cytokine levels revealed a significant decrease in IL-6 in purified compared to non-purified products. RNA sequencing analysis revealed significant differential gene expression profile between CAR-pos and CAR-neg cells, the prior exhibiting an upregulation of genes associated with immune checkpoint regulation, cytokine-cytokine receptor interaction and Th1/Th2 and Th17 cell differentiation pathways.
CAR-neg T cells exhibit a significantly greater expansion compared to CAR-pos T cells alongside CAR-T cell production. The percentage of CAR-neg cells influences the specific cytotoxicity of the product and on the cytokine pattern, which might influence both efficacy and toxicity and, therefore, the number of CAR-neg T cells within the infusion product must be considered in the clinical setting. Purification of CAR-pos T cells could potentially optimize product characteristics.
尽管嵌合抗原受体T细胞（CAR-T）疗法取得了令人鼓舞的成果，但仍有相当一部分患者由于无反应、疾病进展或治疗相关毒性而经历治疗失败。我们的研究旨在探讨CAR-T细胞产品特性，特别是输注产品中CAR阴性细胞的百分比如何影响其疗效和安全性。
在使用白细胞介素-2（IL-2）或IL-7/IL-15进行体外扩增过程中，对CAR阳性（CAR-pos）和CAR阴性（CAR-neg）细胞进行分析。对通过荧光激活细胞分选（FACS）分选的细胞群体评估其细胞毒性作用和细胞因子分泌情况。对CAR-pos和CAR-neg细胞群体进行转录组分析。
在整个体外细胞培养过程中，观察到转导（CAR-pos）T细胞的百分比显著降低，与CAR-pos T细胞相比，无论使用IL-2还是IL-7/IL-15，CAR-neg T细胞在该过程中均表现出更大的扩增。CAR-pos T细胞表现出激活和耗竭标志物表达增加。CAR-pos和CAR-neg的比较分析显示，与未纯化产品相比，100%CAR-pos T细胞的纯化样品具有增强的细胞毒性作用。细胞因子水平定量显示，与未纯化产品相比，纯化产品中IL-6显著降低。RNA测序分析揭示了CAR-pos和CAR-neg细胞之间显著的差异基因表达谱，前者表现出与免疫检查点调节、细胞因子-细胞因子受体相互作用以及Th1/Th2和Th17细胞分化途径相关基因的上调。
与CAR-T细胞生产过程中的CAR-pos T细胞相比，CAR-neg T细胞表现出显著更大的扩增。CAR-neg细胞的百分比会影响产品的特异性细胞毒性和细胞因子模式，这可能会影响疗效和毒性，因此在临床环境中必须考虑输注产品中CAR-neg T细胞的数量。纯化CAR-pos T细胞可能会优化产品特性。
