背景:急性髓性白血病(AML)是一种克隆性恶性疾病,起源于少量的白血病起始细胞或白血病干细胞(LSCs)-亚群也是复发/难治性AML的根本原因。嵌合抗原受体(CAR)-T细胞疗法已被证明在对抗某些血液系统恶性肿瘤方面是成功的。但是有几个障碍限制了它的广泛应用。CAR-自然杀伤(NK)细胞不携带诱导移植物抗宿主病(GvHD)的风险,经常与同种异体T细胞相关,从而克服耗时,自体细胞制造,并且比CAR-T细胞具有相对更安全的临床特征。本研究旨在从用第三代抗TIM3CAR工程化的克隆主诱导多能干细胞中产生靶向LSCs的抗TIM3CAR-NK细胞。
方法:克隆母脐血NK来源的诱导多能干细胞(iPSC)系,MUSIi013-A,用作起始细胞,用于工程化带有TIM3scFv片段的抗TIM3CAR(克隆TSR-022),CD28,4-1BB,和CD3ζ信号(CAR-TIM3)。将建立的CAR-TIM3iPSC在无血清和无饲养细胞条件下进一步分化为功能性CAR-TIM3NK细胞,并测试其针对各种TIM3阳性AML细胞的抗肿瘤活性。
结果:我们成功建立了CAR-TIM3iPSCs的单细胞克隆,通过基因组DNA测序以及抗体和抗原特异性检测验证。我们进行了彻底的iPSC表征以确认其保留的多能性和分化能力。建立的CAR-TIM3iPSC可以分化为CAR-TIM3NK样细胞,与来自亲本iPSC的野生型(WT)NK样细胞相比,进一步证明其对TIM3阳性AML细胞的抗肿瘤活性增强,对TIM3阴性细胞的影响最小。
结论:用CAR工程化的iPSC,包括本文建立的单细胞克隆CAR-TIM3iPSC,是用于产生现成的CAR-NK细胞以及其他CAR免疫细胞的潜在替代细胞来源。功能性CAR-TIM3NK细胞在无血清和无饲养条件下分化的可行性支持良好生产规范(GMP)兼容方案可进一步建立用于未来临床应用。
BACKGROUND: Acute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)-the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells. The present study aimed to generate anti-TIM3 CAR-NK cells targeting LSCs from a clonal master induced pluripotent stem cells engineered with the third-generation anti-TIM3 CAR.
METHODS: A clonal master umbilical cord blood NK-derived induced pluripotent stem cell (iPSC) line, MUSIi013-A, was used as a starting cells for engineering of an anti-TIM3 CAR harboring TIM3 scFv fragment (clone TSR-022), CD28, 4-1BB, and CD3ζ signaling (CAR-TIM3). The established CAR-TIM3 iPSCs were further differentiated under serum- and feeder-free conditions into functional CAR-TIM3 NK cells and tested for its anti-tumor activity against various TIM3-positive AML cells.
RESULTS: We successfully established a single-cell clone of CAR-TIM3 iPSCs, as validated by genomic DNA sequencing as well as antibody and antigen-specific detection. We performed thorough iPSC characterization to confirm its retained pluripotency and differentiation capacity. The established CAR-TIM3 iPSCs can be differentiated into CAR-TIM3 NK-like cells, which were further proven to have enhanced anti-tumor activity against TIM3-positive AML cells with minimal effect on TIM3-negative cells when compared with wild-type (WT) NK-like cells from parental iPSCs.
CONCLUSIONS: iPSCs engineered with CARs, including the established single-cell clone CAR-TIM3 iPSCs herein, are potential alternative cell source for generating off-the-shelf CAR-NK cells as well as other CAR-immune cells. The feasibility of differentiation of functional CAR-TIM3 NK cells under serum- and feeder-free conditions support that Good Manufacturing Practice (GMP)-compliant protocols can be further established for future clinical applications.