Department of Pediatrics, Kyoto Prefectural University of Medicine, Graduate School of Medical Science, Kyoto, Japan.
AGC Inc. Innovative Technology Laboratories, Yokohama, Japan.
Front Immunol. 2022 Jan 27;13:770132. doi: 10.3389/fimmu.2022.770132. eCollection 2022.
The quality of chimeric antigen receptor (CAR)-T cell products, namely, memory and exhaustion markers, affects the long-term functionality of CAR-T cells. We previously reported that (PB) transposon-mediated CD19 CAR-T cells exhibit a memory-rich phenotype that is characterized by the high proportion of CD45RA/C-C chemokine receptor type 7 (CCR7) T-cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA and CD45RA peripheral blood mononuclear cells (PBMCs) (RA CAR and RA CAR, respectively), and compared their phenotypes and antitumor activity. RA CAR-T cells showed better transient gene transfer efficiency 24 h after transduction and superior expansion capacity after 14 days of culture than those shown by RA CAR-T cells. RA CAR-T cells exhibited dominant CD8 expression, decreased expression of the exhaustion marker programmed cell death protein-1 (PD-1) and T-cell senescence marker CD57, and enriched naïve/stem cell memory fraction, which are associated with the longevity of CAR-T cells. Transcriptome analysis showed that canonical exhaustion markers were downregulated in RA CAR-T, even after antigen stimulation. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, the expression of CAR molecules on cell surface after antigen stimulation in RA CAR-T cells was controlled at a relatively lower level than that in RA CAR-T cells. In the stress test, RA CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells compared with RA CAR-T cells. CAR-T cells were not detected in the control or RA CAR-T cells but RA CAR-T cells were expanded even after 50 days of treatment, as confirmed by sequential bone marrow aspiration. Our results suggest that PB-mediated RA CAR-T cells exhibit a memory-rich phenotype and superior antitumor function, thus CD45RA PBMCs might be considered an efficient starting material for PB-CAR-T cell manufacturing. This novel approach will be beneficial for effective treatment of B cell malignancies.
嵌合抗原受体 (CAR)-T 细胞产品的质量，即记忆和衰竭标志物，会影响 CAR-T 细胞的长期功能。我们之前报道过，（PB）转座子介导的 CD19 CAR-T 细胞表现出富含记忆的表型，其特征是 CD45RA/C-C 趋化因子受体 7（CCR7）T 细胞亚群的比例较高。为了进一步研究 PB-CD19 CAR-T 细胞的有利表型，我们从 CD45RA 和 CD45RA 外周血单核细胞（PBMC）中生成 PB-CD19 CAR-T 细胞（RA CAR 和 RA CAR，分别），并比较它们的表型和抗肿瘤活性。RA CAR-T 细胞在转导后 24 小时显示出更好的瞬时基因转移效率，在培养 14 天后显示出更好的扩增能力。RA CAR-T 细胞表现出主导性 CD8 表达、衰竭标志物程序性细胞死亡蛋白-1（PD-1）和 T 细胞衰老标志物 CD57 的表达减少，以及幼稚/干细胞记忆亚群的富集，这些都与 CAR-T 细胞的寿命有关。转录组分析显示，即使在抗原刺激后，RA CAR-T 中的经典衰竭标志物也被下调。虽然抗原刺激可以增加 CAR 的表达，导致持续的 CAR 信号和衰竭，但在 RA CAR-T 细胞中，抗原刺激后细胞表面 CAR 分子的表达水平被控制在相对较低的水平，低于 RA CAR-T 细胞。在应激测试中，RA CAR-T 细胞通过 CAR-T 细胞的扩增实现了对肿瘤的长期控制，优于 RA CAR-T 细胞。在对照或 RA CAR-T 细胞中未检测到 CAR-T 细胞，但即使在治疗 50 天后仍扩增了 RA CAR-T 细胞，这通过连续骨髓抽吸得到了证实。我们的结果表明，PB 介导的 RA CAR-T 细胞表现出富含记忆的表型和优越的抗肿瘤功能，因此 CD45RA PBMC 可能被视为 PB-CAR-T 细胞制造的有效起始材料。这种新方法将有利于有效治疗 B 细胞恶性肿瘤。
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