Expression analysis of chimeric antigen receptor-transduced T cells following antigenic stimulation. Homo sapiens

Adoptive transfer of anti-CD19 chimeric antigen receptor (CAR)-engineered T cells has shown impressive clinical responses in patients with refractory B-cell malignancies. However, therapeutic effects of CAR-T cells targeting other hematologic malignancies and solid tumors are not yet satisfactory. Although inefficient tumor trafficking and multiple immunosuppressive molecules impede CAR-T cell effector responses, signals delivered by the current CAR constructs may still be insufficient to fully activate antitumor T cell functions. Optimal T cell activation and proliferation requires multiple signals including T cell receptor (TCR) engagement (signal 1), costimulation (signal 2), and cytokine engagement (signal 3). CAR genes developed to date contain a CD3z domain and costimulatory domain(s), but not a domain to transmit signal 3. In this study, we have developed a novel CAR construct capable of inducing cytokine signaling in an antigen-dependent manner. The new generation CD19 CAR encodes a cytoplasmic domain of IL-2RB and STAT3-binding YXXQ motif together with CD3z and CD28 domains (28-IL2RB-z (YXXQ)). The 28-IL2RB-z (YXXQ) CAR-T cells showed antigen-dependent JAK-STAT, especially the STAT3-mediated pathway activation, which promoted their proliferation and prevented terminal differentiation. The 28-IL2RB-z (YXXQ) CAR-T cells demonstrated superior in vivo persistence and antileukemia effects compared with the currently used CARs in multiple tumor models. Overall design: Human CD8+ T cells derived from three different healthy donors were retrovirally transduced with individual anti-CD19 CAR genes (28-z, 28-BB-z and 28-IL2RB-z (YXXQ)) and were stimulated with K562 cells expressing CD19. RNA was collected 4 days after stimulation and gene expression profiles were analyzed by Affymetrix Human Gene 2.0 ST Array (gene-level analysis).

抗CD19嵌合抗原受体（chimeric antigen receptor, CAR）工程化T细胞的过继转移疗法，在难治性B细胞恶性肿瘤患者中已展现出显著的临床应答。然而，靶向其他血液系统恶性肿瘤与实体瘤的CAR-T细胞治疗效果仍不尽人意。尽管低效的肿瘤归巢与多种免疫抑制分子会阻碍CAR-T细胞的效应功能，但现有CAR结构传递的信号或许仍不足以完全激活抗肿瘤T细胞的功能。
最佳的T细胞活化与增殖需要多重信号，包括T细胞受体（T cell receptor, TCR）结合（第一信号）、共刺激（第二信号）以及细胞因子结合（第三信号）。迄今为止开发的CAR基因仅包含CD3ζ结构域与共刺激结构域，却缺乏能够传递第三信号的结构域。
本研究中，我们开发了一种新型CAR结构，可通过抗原依赖性方式诱导细胞因子信号转导。新一代CD19 CAR携带有白细胞介素2受体β链（IL-2RB）胞质结构域与信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3）结合型YXXQ基序，并同时搭载CD3ζ与CD28结构域（命名为28-IL2RB-z (YXXQ)）。28-IL2RB-z (YXXQ) CAR-T细胞可展现出抗原依赖性的贾纳斯激酶-信号转导与转录激活因子（JAK-STAT）通路活化，尤其是STAT3介导的信号通路，该效应可促进其增殖并阻止细胞终末分化。相较于当前使用的CAR结构，28-IL2RB-z (YXXQ) CAR-T细胞在多种肿瘤模型中展现出更优异的体内存续能力与抗白血病效应。
整体实验设计：从3名健康供者中分离得到人CD8+ T细胞，使用三种不同的抗CD19 CAR基因（28-z、28-BB-z与28-IL2RB-z (YXXQ)）进行逆转录病毒转导，随后用表达CD19的K562细胞进行刺激。刺激4天后收集RNA样本，通过Affymetrix人类基因2.0 ST芯片完成基因表达谱分析（基因水平分析）。