CXCR4-Expressing Anti-CD25 CAR T-Cells Effectively Eliminate Human AML Cells In Vivo-猫眼课题宝

DOI：

10.1182/blood-2020-142228

发表时间：

2020-11

期刊：

Blood

影响因子：

20.3

通讯作者：

中科院分区：

医学1区

文献类型：

作者：

Ari Itoh-Nakadai;Yoriko Saito;Mariko Murasawa-Tomizawa;H. Kajita;Takehisa Matsumoto;Masashi Matsuda;Takashi Watanabe;M. Shirouzu;O. Ohara;H. Koseki;L. Shultz;F. Ishikawa

靶向CD 19的嵌合抗原受体（CAR）T细胞已成为复发性/难治性B细胞急性淋巴细胞白血病和弥漫性大B细胞淋巴瘤的有希望的治疗选择。对于急性髓性白血病（AML），靶向分子如CD 33和CD 123的CAR T细胞正在进行临床评估。无论靶向疾病或抗原如何，都必须了解CAR T细胞的靶向和脱靶效应的机制，如细胞因子释放综合征。在这项研究中，我们的目标是开发一种CAR T细胞治疗预后不良的AML。为此，我们在NOD/SCID/Il 2 rgKO（NSG）异种移植试验中分析了具有体内AML发展能力的患者来源AML起始细胞的基因表达。与正常的CD 34 + CD 38-造血干/祖细胞（HSPC）相比，先前报道为AML中不良预后的标志物的CD 25（IL-2受体α链）在AML起始细胞中过度表达（Saito et al.，Science Translational Medicine 2010）。此外，该抗原在其他血液恶性肿瘤如CML、成人T细胞白血病/淋巴瘤和霍奇金淋巴瘤中表达。因此，我们设计了含有TCR序列和人CD 25抗原的Fab抗原识别位点的慢病毒载体。在将⑶ 25-CAR慢病毒颗粒转导到脐带血来源的人T细胞中之后，我们实现了体外⑶ 25-CAR T细胞扩增至超过2xl 07个细胞。用5x 106个CD 25-CAR（25 CAR）T细胞（患者n=3，每个患者PDX n=3）体内治疗人AML患者来源的异种移植（PDX）小鼠导致PDX小鼠的外周血（PB）中患者来源的白血病细胞减少，但骨髓（BM）中仍有大量白血病细胞。为了改善骨髓中AML细胞的归巢和靶向，我们工程化了具有小鼠CXCR 4表达的CAR构建体（CXCR 4 - 25 CAR）。注射5xl 06个CXCR 4 - 25 CAR T细胞导致PDX小鼠PB中人AML细胞的完全消除0 +/-0.0 hCD 33+细胞/ml，n=3），而患者来源的AML细胞保留在用5xl 06个非CXCR 4表达的25 CAR T细胞处理的PDX的外周血中。（1416.2+/-661.0 hCD 33+细胞/ml PB，n=3）（未处理PDX：15677 hCD 33+细胞/ml PB，n=1）。在CAR T细胞注射后4周，我们发现仅在用CXCR 4 - 25 CAR T治疗的小鼠中hCD 33 + AML细胞完全根除。（CXCR 4 - 25 CAR T细胞处理：BM 0.0+/-0.0细胞，脾0.0+/-0.0细胞，脾，n=3; 25 CAR T细胞处理：BM 1.9+/-0.4x107细胞，脾6.2+/-2.8x107细胞，n=3）。此外，组织病理学检查证明在CXCR 4 25 CAR处理的小鼠的肝、肺和肠中没有异种GVHD。在一只经CXCR 4 - 25 CAR T细胞治疗的PDX小鼠中，经长期观察，PB中CXCR 4 -25 CAR T细胞的数量减少，鼠CD 45+造血细胞增加，3个月后无AML复发的证据（注射后2周：hCD 33 +AML 1040.3 +/-159.7个细胞/ml，hCD 3 +T细胞474.7 +/-85.8个细胞/ml，小鼠CD 45+细胞751 +/-31.0个细胞/ml，n=3;注射后3个月：hCD 33 +AML 0.0+/-0.0个细胞/ml，hCD 3 +T细胞136.3 +/-77.6个细胞/ml，mCD 45+细胞1864 +/-428个细胞/ml，n=3）。这些发现表明，人AML细胞的消除是由CAR T细胞特异性靶向CD 25介导的。在移植有正常CB CD 34 + CD 38-HPSC的NSG小鼠中注射表达CXCR 4的CD 25-CAR T细胞，人CD 3 + Foxp 3+细胞在PB CD 4 + T细胞中的百分比没有变化（注射前10.5+/-1.4%，n=2;注射后4周：14.8+/-0.9%，n=2）。此外，CXCR 4 - 25 CAR对PB、BM和脾脏中的CD 3 + CD 4 + FoxP 3+细胞、总CD 3+细胞、CD 19+细胞、CD 33+细胞和CD 56+细胞的细胞数量没有影响。表达CXCR 4的CD 25-CAR T细胞是预后不良AML的一种有前景的治疗策略。无相关利益冲突需要声明。

Chimeric antigen receptor (CAR) T-cells targeting CD19 has become a promising treatment option for relapsed/refractory B cell acute lymphoblastic leukemia and diffuse large B Cell lymphoma. For acute myeloid leukemia (AML), CAR T-cells targeting molecules such as CD33 and CD123 are under clinical evaluation. Regardless of target diseases or antigens, it is essential to understand mechanisms underlying both on- and off-target effects of CAR T-cells such as cytokine release syndrome. In the study, we aimed to develop a CAR T-cell treatment for poor prognosis AML. To this end, we analyzed gene expression of patient-derived AML-initiating cells with demonstrated capacity for in vivo AML development in a NOD/SCID/Il2rgKO (NSG) xenogeneic transplantation assay. CD25 (IL-2 receptor alpha chain), previously reported as a marker for poor prognosis in AML, was over-represented in AML-initiating cells as compared with normal CD34+CD38- hematopoietic stem/progenitor cells (HSPCs) (Saito et al., Science Translational Medicine 2010). In addition, the antigen is expressed in other hematologic malignancies such as CML, adult T cell leukemia/lymphoma, and Hodgkin's lymphoma. We therefore engineered lentiviral vector containing TCR sequence and Fab antigen recognition sites for human CD25 antigen. Following transduction of CD25-CAR lentiviral particles into cord blood-derived human T cells, we achieved in vitro CD25-CAR T-cell expansion to more than 2x107 cells. In vivo treatment of human AML patient-derived xenotransplantation (PDX) mice with 5x106 CD25-CAR (25CAR) T-cells (patient n=3, PDX n=3 for each patient) resulted in reduction of patient-derived leukemic cells in the peripheral blood (PB) of PDX mice, but abundant leukemic cells remained in the bone marrow (BM). To improve homing and targeting of AML cells in the BM, we engineered CAR construct with mouse CXCR4 expression (CXCR4-25CAR). Injection of 5x106 CXCR4-25CAR T-cells resulted in complete elimination of human AML cells in PB of PDX mice (0.0+/-0.0 hCD33+ cells/ml, n=3), while patient-derived AML cells remained in peripheral blood of PDX treated with 5x106 non-CXCR4-expressing 25CAR T-cells (1416.2+/-661.0 hCD33+ cells/ml PB, n=3) (Untreated PDX: 15677 hCD33+ cells/ml PB, n=1). At 4 weeks post-CAR T-cell injection, we found complete eradication of hCD33+ AML cells only in mice treated with CXCR4-25CAR T (CXCR4-25CAR T-cell treated: BM 0.0+/-0.0 cells, spleen 0.0+/-0.0 cells spleen, n=3; 25CAR T-cell treated: BM 1.9+/-0.4x107 cells, spleen 6.2+/-2.8x107 cells, n=3). In addition, histopathological examination demonstrated no xenogeneic GVHD in liver, lung, and intestine of the CXCR4 25 CAR-treated mice. In one CXCR4-25CAR T-cell-treated PDX mouse with longer-term observation, the number of CXCR4-25 CAR T-cells decreased and murine CD45+ hematopoietic cells increased in PB without evidence of AML relapse after 3 months (2 weeks post-injection: hCD33+AML 1040.3 +/-159.7 cells/ml, hCD3+T cells 474.7 +/-85.8 cells/ml, mouse CD45+ cells 751 +/-31.0 cells/ml, n=3; 3 months post-injection: hCD33+AML 0.0+/-0.0 cells/ml, hCD3+T cells 136.3 +/-77.6 cells/ml, mCD45+ cells 1864 +/-428 cells/ml, n=3). These findings indicate that elimination of human AML cells is mediated by specific targeting of CD25 by CAR T-cells. Injection of CXCR4-expressing CD25-CAR T-cells in NSG mice engrafted with normal CB CD34+CD38- HPSCs resulted in no change in the percentage of human CD3+Foxp3+ cells among PB CD4+ T cells (Pre-injection 10.5+/-1.4%, n=2; 4 weeks post-injection: 14.8+/-0.9% n=2). Additionally, CXCR4-25CAR did not affect to cell numbers of CD3+CD4+FoxP3+ cells, total CD3+ cells, CD19+ cells, CD33+ cells, and CD56+ cells in PB, BM, and spleen. CXCR4-expressing CD25-CAR T-cells is a promising treatment strategy for poor prognosis AML. No relevant conflicts of interest to declare.

CXCR4-Expressing Anti-CD25 CAR T-Cells Effectively Eliminate Human AML Cells In Vivo

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