Generation of TCR-negative CD8αβ+ CAR T cells from T cell-derived induced pluripotent stem cells

The production of autologous T cells expressing a chimaeric antigen receptor (CAR) is time-consuming, costly and occasionally unsuccessful. T cell-derived induced pluripotent stem cells are a promising source for the generation of ‘off-the-shelf’ CAR T cells, but their in vitro differentiation often yields T cells with suboptimal features. Here we show that premature expression of the T cell receptor (TCR) or a constitutively expressed CAR promote the acquisition of an innate phenotype, which can be averted by disabling the TCR and relying on the CAR to drive T cell differentiation. Delaying CAR expression and calibrating its signalling strength enabled the generation of human CD8αβ+ TCR– CAR+ T cells that perform overall similarly to peripheral blood CD8αβ+ CAR+ T cells in achieving effective tumour control upon systemic administration in a mouse model of leukaemia, without causing graft-versus-host disease. Driving T cell maturation in the absence of a TCR by taking advantage of a CAR may facilitate the large-scale development of potent allogeneic CD8αβ+ T cells for a broad range of immunotherapies. TRAC-1XX-iT cell generation: TiPS were differentiated to the DP T cell stage, and matured to CD8ab SP cells on 3T3-CD19-41BBL. CD8ab SP cells were purified by flow cytometry. Peripheral Blood Mononuclear cells were purified from healthy donor whole blood. CD4 and CD8 T cells were targeted with CD19-28z-1XX CAR into the TRAC locus, NK and gdT cells were retrovirally transduced to express the CD19-28z-1XX CAR. Cells were purified for CAR expression by flow cytometry. mRNA profiles of PBMC-derived ab-TCR CD8ab T cells, gd-TCR T cells, CD8ab TRAC-1XX-iT cells.

表达嵌合抗原受体（chimaeric antigen receptor, CAR）的自体T细胞的制备流程耗时冗长、成本高昂，且偶有失败。源自T细胞的诱导多能干细胞是制备现货型CAR-T细胞的极具潜力的候选来源，但其体外分化所得的T细胞往往存在功能特性不佳的问题。本研究证实，T细胞受体（T cell receptor, TCR）的过早表达或组成型表达的CAR会诱导细胞获得先天表型，而通过失活TCR并依托CAR驱动T细胞分化，可有效规避这一缺陷。延迟CAR表达并校准其信号强度，能够获得人CD8αβ+ TCR阴性CAR阳性T细胞；在白血病小鼠模型中经系统给药后，这类细胞的肿瘤控制效果与外周血来源的CD8αβ+ CAR+ T细胞整体相当，且不会引发移植物抗宿主病（graft-versus-host disease, GVHD）。借助CAR在无TCR的条件下驱动T细胞成熟，可为大规模制备适用于多种免疫治疗的高效同种异体CD8αβ+ T细胞提供有力支撑。TRAC-1XX-iT细胞的制备：将T细胞来源诱导多能干细胞（TiPS）分化至双阳性T细胞阶段，并在3T3-CD19-41BBL细胞上诱导成熟为CD8αβ单阳性T细胞，随后通过流式细胞术纯化得到目标细胞。从健康供者全血中分离纯化外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMC）；将CD19-28z-1XX CAR靶向整合至TRAC基因座，以转导CD4及CD8 T细胞；通过逆转录病毒转导使NK细胞与γδT细胞表达CD19-28z-1XX CAR。最后通过流式细胞术筛选纯化表达CAR的细胞。本数据集的转录组表达谱涵盖PBMC来源的αβTCR CD8αβ+ T细胞、γδTCR T细胞以及CD8αβ TRAC-1XX-iT细胞。