免疫检查点的治疗性干扰显著推进了治疗癌症的方法。程序性死亡-1（PD-1）/程序性死亡配体-1轴在下调T细胞功能方面发挥着显著作用，为增强CAR-T细胞疗法的疾病修改作用提供了一个可行的策略。为了处理检查点干扰，使用下一代基于CRISPR的平台（Cas9 chRDNA）对原代人类T细胞进行基因组编辑，通过敲除编码PD-1受体的PDCD1基因来实现。将特异性针对CD19的嵌合抗原受体位点特异性插入到T细胞受体α常量基因座中，以推动细胞毒性活性。这些异基因CAR-T细胞（CB-010）与基因相同但没有PDCD1敲除的CAR-T细胞相比，可促进小鼠在B细胞恶性肿瘤的已建立的原位肿瘤异种移植模型中更长时间的生存。CB-010的持久动力学与没有PDCD1干扰的CAR-T细胞在血液学组织中相似，表明建立的肿瘤异种移植的强大初始减负是由于增强了机能适应性。通过单细胞RNA-Seq分析，与没有PDCD1敲除的基因工程CAR-T细胞相比，CB-010细胞表现出更少的调节性T细胞，更低的疲劳表型和减少的功能障碍标记，并具有更高的激活、糖酵解和氧化磷酸化标记。此外，观察到线粒体代谢适应性增强，包括增加的呼吸能力，也是未分化T细胞的标志。在同源异基因CAR-T细胞疗法的背景下，基因组PD-1检查点干扰可能为治疗B淋巴细胞恶性肿瘤提供一个引人注目的选择。版权所有©2023国际细胞与基因治疗学会。由Elsevier Inc.出版。保留所有权利。

Therapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy.To address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity.These allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells.Genomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.