Objective: To explore a novel strategy that addresses the dual challenges of fratricide and on-target off-tumor toxicity in current chimeric antigen receptor T-cell (CAR-T) therapy for T-cell acute lymphoblastic leukemia (T-ALL) and to develop a safe and efficacious anti-T-ALL CAR-T product by identifying a new target and compatible gene-editing approach. Methods: Public single-cell RNA sequencing (scRNA-seq) datasets were utilized to analyze bone marrow cells extracted from patients with T-ALL and healthy donors, evaluating the differential expression profiles of CD6 and CD7. In investigating the endogenous role of CD6 in CAR-T cells, the CRISPR/Cas9 RNP system was first employed in a CD19 CAR-T model to evaluate the impact of CD6 knockout on the phenotype and activation status of CAR-T cells. Subsequently, CD6-knockout, CD6-targeted CAR-T cells (6KO-6CAR) were constructed, and their functional activities were evaluated. Results: scRNA-seq analysis revealed that CD6 is broadly expressed in T-ALL. Compared with the traditional target CD7, which is also expressed in a subset of normal hematopoietic stem/progenitor cells and myeloid cells, CD6 exhibits a more restricted expression profile, exhibiting superior safety characteristics. Studies on the CD19 CAR-T model indicated that CD6 knockout enables CAR-T cells to maintain a superior functional state: their baseline activation level (CD25 expression) was reduced (P<0.05) while generating a higher proportion of TNF-α(+)IFN-γ(+) cells (P<0.05) upon antigen stimulation. The further constructed 6KO-6CAR cells exhibited potent specific activation (significantly upregulated CD107a expression level, all P<0.001) and cytotoxicity (all P<0.05) against multiple CD6(+) T-ALL cell lines (MOLT-4, CCRF-CEM, and Jurkat) in vitro. Conclusion: CD6 is a novel therapeutic target for T-ALL with high coverage and a favorable safety profile, and knocking out endogenous CD6 globally optimizes the intrinsic functional state of CAR-T cells. Constructing 6KO-6CAR based on the CRISPR/Cas9 technology addresses fratricide in CAR-T cells while enhancing their antitumor functionality, thereby providing a novel immunotherapy regimen with safety and clinical translational potential for relapsed/refractory T-ALL. 目的： 探索克服急性T淋巴细胞白血病（T-ALL）嵌合抗原受体T细胞（CAR-T细胞）疗法中自相残杀与非肿瘤靶向毒性双重障碍的新策略，通过鉴定新靶点及其相应基因编辑方案，开发一种安全、高效的抗T-ALL CAR-T产品。 方法： ①利用公共数据库单细胞转录组测序（scRNA-seq）数据分析T-ALL患者及健康供者骨髓细胞，评估CD6与CD7的表达谱差异。②为探究CD6在CAR-T细胞中的内源性作用，首先采用CRISPR/Cas9 RNP系统在CD19 CAR-T模型中，评估CD6敲除对CAR-T细胞表型和活化状态的影响；随后，构建CD6敲除的靶向CD6 CAR-T细胞（6KO-6CAR）并评估其功能。 结果： scRNA-seq数据显示，CD6在T-ALL中广泛表达，且相较于在部分正常造血干/祖细胞及髓系细胞中均表达的传统靶点CD7，CD6的表达谱更为局限，展现出更优的安全性特征。CD19 CAR-T模型中的研究表明，CD6敲除能使CAR-T细胞维持更优的功能状态：其基线活化水平（CD25表达）降低（P<0.05），同时在抗原刺激下能产生更高比例的TNF-α(+)IFN-γ(+)细胞（P<0.05）。进一步构建的6KO-6CAR细胞在体外对多种CD6(+) T-ALL细胞系（MOLT-4、CCRF-CEM、Jurkat）表现出较强的特异性激活（CD107a表达显著上调，均P<0.001）及杀伤作用（均P<0.05）。 结论： CD6是一个具有高覆盖性和良好安全性的T-ALL治疗新靶点，同时敲除内源性CD6后还能从整体上优化CAR-T细胞的内在功能状态。基于CRISPR/Cas9技术构建6KO-6CAR的策略，能在解决CAR-T细胞自相残杀的同时增强其抗肿瘤功能，这为T-ALL提供了一种兼具安全性与应用前景的新型免疫治疗方案。.
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PubMed · 2026-05-14
PubMed · 2026-04-14
PubMed · 2026-04-14
PubMed · 2026-05-14