Abstract：
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.
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.
摘要：
目的：对免疫检查点的治疗性破坏显著推进了癌症治疗方法的进展。程序性死亡-1（PD-1）/程序性死亡配体-1轴下调T细胞功能的突出作用为增强CAR-T细胞疗法的疾病修饰作用提供了一种易于处理的策略。
方法：要解决检查点干扰，通过敲除编码PD-1受体的PDCD1基因,使用下一代基于CRISPR的平台(Cas9chRDNA)对原代人T细胞进行基因组编辑.实施对CD19具有特异性的嵌合抗原受体到T细胞受体α恒定基因座中的位点特异性插入以驱动细胞毒性活性。
结果：这些同种异体CAR-T细胞（CB-010）与没有PDCD1敲除的相同工程化CAR-T细胞相比，在建立良好的B细胞恶性肿瘤原位肿瘤异种移植模型中，小鼠的生存期更长。血液组织中CB-010细胞的持久性动力学与没有PDCD1破坏的CAR-T细胞相似，表明建立的肿瘤异种移植物的稳健初始减积是由于功能适应性增强。通过单细胞RNA-Seq分析，CB-010单元,与没有PDCD1敲除的相同工程CAR-T细胞相比，表现出更少的Treg细胞，较低的耗竭表型和减少的功能障碍特征，并具有较高的激活，糖酵解和氧化磷酸化特征。Further,观察到线粒体代谢适应性的增强，包括增加呼吸能力，分化较低的T细胞的标志。
结论：在同种异体CAR-T细胞治疗的背景下，基因组PD-1检查点破坏可能为治疗B淋巴恶性肿瘤提供了一个令人信服的选择。
方法：要解决检查点干扰，通过敲除编码PD-1受体的PDCD1基因,使用下一代基于CRISPR的平台(Cas9chRDNA)对原代人T细胞进行基因组编辑.实施对CD19具有特异性的嵌合抗原受体到T细胞受体α恒定基因座中的位点特异性插入以驱动细胞毒性活性。
结果：这些同种异体CAR-T细胞（CB-010）与没有PDCD1敲除的相同工程化CAR-T细胞相比，在建立良好的B细胞恶性肿瘤原位肿瘤异种移植模型中，小鼠的生存期更长。血液组织中CB-010细胞的持久性动力学与没有PDCD1破坏的CAR-T细胞相似，表明建立的肿瘤异种移植物的稳健初始减积是由于功能适应性增强。通过单细胞RNA-Seq分析，CB-010单元,与没有PDCD1敲除的相同工程CAR-T细胞相比，表现出更少的Treg细胞，较低的耗竭表型和减少的功能障碍特征，并具有较高的激活，糖酵解和氧化磷酸化特征。Further,观察到线粒体代谢适应性的增强，包括增加呼吸能力，分化较低的T细胞的标志。
结论：在同种异体CAR-T细胞治疗的背景下，基因组PD-1检查点破坏可能为治疗B淋巴恶性肿瘤提供了一个令人信服的选择。
