CAR-T细胞治疗在急性髓系白血病中的研究和应用进展

急性髓系白血病（acute myeloid leukemia,AML）是一种高度异质性的恶性血液病,近些年随着化疗、靶向药物和造血干细胞移植的发展,AML患者的疗效已有较大的提高,但总体而言,绝大部分患者仍无法治愈。目前,成人AML患者5年生存率仍未超过30%。嵌合抗原受体T细胞（chimeric antigen receptor T-cell,CAR-T）疗法在复发难治性B淋巴细胞肿瘤领域取得了显著疗效,越来越多的研究开始研发CAR-T疗法在其他类型肿瘤及疾病中的应用。本文主要就近些年CAR-T疗法在AML中的临床应用和研究进展做出综述。      

靶向CD33 的CAR修饰的NK92 细胞对CD33+急性髓系白血病细胞的杀伤作用

[摘要] 目的：根据抗CD33-scFv 序列构建CD33-CAR 修饰的NK92 细胞,探讨其对CD33+ 急性髓系白血病（acute myeloidleukemia,AML）细胞的杀伤作用。方法：通过基因合成以及分子克隆技术获得抗CD33-CAR片段,然后将其构建到慢病毒载体上,进行慢病毒包装,将得到的慢病毒转染NK92 细胞,用流式细胞术检测细胞转染效率并通过嘌呤霉素筛选得到稳定表达抗CD33-CAR的细胞系CD33-CAR-NK92,利用钙黄绿素释放法检测该细胞的杀伤作用,ELISA法检测细胞因子IFN-γ 分泌的变化。结果：成功构建pCDH-CD33-CAR 重组慢病毒质粒,慢病毒转导后约18.7%的NK92 细胞表达CD33-CAR（命名为CD33-CARNK92细胞）,嘌呤霉素筛选后表达CD33-CAR的NK92 细胞比例约86.3%。CD33-CAR-NK92 细胞对CD33+AML细胞MOLM-13的杀伤作用明显高于未被基因修饰的NK92 细胞（P<0.01）,而两者对CD33-肿瘤细胞JURKAT 的杀伤作用没有明显差异（P>0.05）。效靶比为2∶1 共培养6 h 后,经CD33-CAR修饰的NK92 细胞相比未被基因修饰的NK92 细胞IFN-γ 的分泌水平明显升高[ （190.97±11.52）vs（88.41±2.75）pg/ml, P<0.01]。结论：CD33-CAR-NK92 细胞能特异性识别CD33 抗原并杀伤CD33+AML细胞,其杀伤作用显著高于未被基因修饰的NK92 细胞,为进一步开展靶向CD33 的CAR-NK92 细胞治疗AML的临床转化奠定了实验基础。     

CD34阳性成人急性髓系白血病临床及生物学研究

检测了４０例成人初治急性髓系白血病细胞ＣＤ３４抗原的表达，并分析了ＣＤ３４表达与一般临床特征，其它免疫标志，。染色体异常及治疗效果的关系。结果显示，４０例ＡＭＬ中ＣＥ３４阳性表达１７例，占４２．５％，除Ｍ３无ＣＤ３４表达外，ＣＤ３４表达与ＦＡＢ亚型无关。     

CD7阳性的急性髓系白血病临床及细胞遗传学特征

 目的 研究CD7在急性髓系白血病（AML）患者中的表达情况,分析CD7阳性AML患者临床特点与细胞遗传学特征。方法 对130例AML患者进行研究,用流式细胞仪检测免疫表型,比较CD7阳性AML和CD7阴性AML患者的性别、年龄、外周血白细胞计数、骨髓原始细胞计数等。同时检测CD7阳性患者的染色体核型。结果 AML患者的CD7阳性率为10 %,CD7阳性AML外周血白细胞计数、骨髓原始细胞计数、染色体异常率均高于CD7阴性者。完全缓解率低,生存期短。CD7阳性AML染色体异常率为71.4 %,预后差核型多见。结论 CD7抗原可作为疾病预后差、复发、恶化的指标,CD7阳性的AML患者预后不良。     

CD4抗原在急性髓系白血病细胞上的表达及其意义

目的：探讨ＣＤ４抗原在急性髓系白血病细胞上的表达及意义。方法：对８２例急性髓系白血病患者进行免疫表型、细胞遗传学分析。结果：ＣＥ４在ＡＭＬ患者中表达率为４０．２％，Ｍ５中阳性率最高９２．９％，Ｍ４次之为５５％，ＣＤ４＾＋ＡＭＬ高表达ＨＬＡ－ＤＲ，ＣＤ３８、ＣＤ３３、ＣＤ１５、ＣＤ１４、ＴＢ系列抗原阴性。ＣＤ４＾＋ＡＭＬ中可见１１ｑ２３、ｉｎｖ（１６）、ｔ（９；２２），未见特异性染色体异常。但伴１１ｑ２３和ｉｎｖ（１６）异常的ＡＭＬ频繁表达ＣＤ４，阳性率分别为８６．３％、６０．２％。ＣＤ４＾＋ＡＭＬ在年龄、性别、肝脾肿大、ＣＮＳ－Ｌ、ＤＩＣ、１疗程ＣＲ率等临床特征方面无明显不同。结论：ＣＤ４＾＋ＡＭＬ是一种起源较高的具有单核细胞特征的髓系白血病，ＣＤ４的表达对ＡＭＬ－Ｍ４、Ｍ５尤其是Ｍ５亚型的鉴别诊断有重要价值。     

抗CD123抗体靶向治疗急性髓系白血病

白血病干细胞（leukemic stem cells,LSCs）被认为是白血病发生、发展、耐药、复发的根源,如何彻底根除LSCs已成为白血病治疗研究的一个重要方向。CD123是LSCs表面相对特异的抗原,针对其研发的靶向抗体治疗药物可有效杀伤急性髓系白血病（acute myeloid leukemia,AML）的LSCs,本文对抗CD123抗体靶向治疗AML的最新进展做一综述。     

吉妥珠单抗在CD33阳性急性髓系白血病中的研究进展

目前,通过运用传统化疗或造血干细胞移植治疗急性髓系白血病（acute myeloid leukemia,AML）虽然取得一定疗效, 但远期预后仍存在局限性。为改善这一缺陷, 抗体偶联药物将可能成为这部分患者的最佳选择。吉妥珠单抗（gemtuzumab ozogamicin,GO）是一种由人源化抗CD33单抗与脱氧核糖核酸（deoxyribonucleic acid,DNA）嵌入剂卡奇霉素（calicheamicin,CLM）形成的抗体偶联药物。CD33表达于90%的AML细胞表面,不表达于正常造血干细胞和成熟粒细胞。因此,其成为AML靶向治疗的良好靶点。已有许多研究报道,GO无论是单药还是联合治疗,均能改善CD33阳性AML患者的预后。本文主要就GO的特征及其在CD33阳性AML中的研究进展进行综述。      

CD19-CAR-T细胞治疗难治复发急性淋巴细胞白血病的研究进展

难治复发急性淋巴细胞白血病（acute lymphocytic leukemia, ALL）预后差,生存期短,其治疗已成为国际难题。嵌合抗原受体基因修饰T（chimeric antigen receptor gene-modified T,CAR-T）细胞是目前最具应用前景的靶向免疫治疗,靶向CD19的CAR-T细胞（CD19-CAR-T）治疗儿童及成人难治复发性ALL的完全缓解率（complete remission,CR）可达90%以上,疗效远高于化疗。然而,细胞因子释放综合征（cytokine release syndrome,CRS）、严重神经毒性（serious neurotoxicity,SNT）、脱靶效应以及疾病复发等严重限制了CAR-T细胞的进一步临床应用。本文主要阐述CAR-T细胞的制备技术、预处理方案、细胞输注剂量及各种并发症防治策略等最新研究进展。     

The clinical significance of soluble CD86 levels in patients with acute myeloid leukemia and myelodysplastic syndrome

BACKGROUND: Levels of the soluble form of CD86 (sCD86) are elevated in a proportion of patients with leukemia. Although it is a potential modulator of antitumor responses, the significance of sCD86 in patients with hematologic malignancies is unknown. METHODS: The authors evaluated sCD86 levels by enzyme-linked immunosorbent assay in patients with acute myeloid leukemia (AML) (n = 57 patients) and patients with myelodysplastic syndrome (MDS) (n = 40 patients) and analyzed the relation between sCD86 levels and various clinical parameters. RESULTS: Levels of sCD86 were elevated (> 2.32 ng/mL) relative to normal donors (0.22-2.32 ng/mL; n = 51 patients) in 25% of patients with AML and in 27% of patients with MDS. Patients with AML who had elevated sCD86 levels had significantly lower complete remission (CR) rates compared with patients with AML who had normal sCD86 levels. In multivariate analysis using sCD86 as a continuous variable and including the interaction of age and sCD86 as a variable, sCD86 was a significant prognostic factor (P = 0.014) independent of cytogenetics. Further analysis demonstrated that, in patients with AML age 60 years and younger, but not in patients older than 60 years, elevated sCD86 levels were associated with significantly shorter survival (P = 0.04). There was no correlation between sCD86 levels and CR rates or survival in patients with MDS. CONCLUSIONS: The presence in patients with AML of elevated levels of circulating sCD86 were associated with lower CR rates and poor survival. The prognostic significance of sCD86 was independent of cytogenetics but was modulated by age, in that it was independently significant only in younger patients. The results suggest that sCD86 may play a role in modulating immune responses associated with the progression of AML.     

吉妥珠单抗在CD33阳性急性髓系白血病中的研究进展

急性髓系白血病（acute myeloid leukemia,AML）是一种异质性髓系恶性肿瘤,目前化疗联合造血干细胞移植是主要治疗方法,但是总体预后较差。吉妥珠单抗（gemtuzumab ozogamicin,GO）是一种人源化抗CD33单抗与卡奇霉素结合的抗体偶联药物,主要用于治疗CD33阳性AML。虽然研究发现GO可以改善CD33阳性AML患者的预后,但是仍有部分AML患者并未获益。GO治疗AML的疗效主要与CD33表达及单核苷酸多态性（single nucleotide polymorphism,SNP）、ATP 结合盒亚家族B成员1（ATP-binding cassette subfamily B member 1,ABCB1）基因及SNP、特异的分子生物学和细胞遗传学等因素有关。本文就GO对AML疗效影响因素的研究进展进行综述。     

Non-cleavable hinge enhances avidity and expansion of CAR-T cells for acute myeloid leukemia

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Chimeric antigen receptor T cells derived from CD7 nanobody exhibit robust antitumor potential against CD7-positive malignancies

The great success of chimeric antigen receptor T (CAR-T)-cell therapy in B-cell malignancies has significantly promoted its rapid expansion to other targets and indications, including T-cell malignancies and acute myeloid leukemia. However, owing to the life-threatening T-cell hypoplasia caused by CD7-CAR-T cells specific cytotoxic against normal T cells, as well as CAR-T cell-fratricide caused by the shared CD7 antigen on the T-cell surface, the clinical application of CD7 as a potential target for CD7⁺ malignancies is lagging. Here, we generated ^CD7ΔT cells using an anti-CD7 nanobody fragment coupled with an endoplasmic reticulum/Golgi retention domain and demonstrated that these cells transduced with CD7-CAR could prevent fratricide and achieve expansion. Additionally, ^CD7ΔCD7-CAR-T cells exhibited robust antitumor potiential against CD7⁺ tumors in vitro as well as in cell-line and patient-derived xenograft models of CD7-positive malignancies. Furthermore, we confirmed that the antitumor activity of CD7-CAR-T cells was positively correlated with the antigen density of tumor cells. This strategy adapts well with current clinical-grade CAR-T-cell manufacturing processes and can be rapidly applied for the therapy of patients with CD7⁺ malignancies.     

FA方案用于急性髓系白血病不同治疗阶段疗效分析

Objective  To evaluate the therapeutic effect of the fludarabine and cytarabine (FA) regimen on acute myeloid leukemia (AML) at different phases during treatment. Methods  A total of 185 patients with AML were divided into 4 groups based on the outcome of previous treatments. Patients in Group 1 had no remission after the first course of induction chemotherapy (n = 55). Patients in Group 2 had no remission after no less than two courses of induction chemotherapy (n = 41). Patients in Group 3 had early relapse (n = 40). Patients in Group 4 had late relapse (n = 49). Patients in groups 2, 3 and 4 had refractory AML or AML with relapse. We assessed the efficacy and toxicity of FA combination chemotherapy in each of these 4 groups. Results  The complete remission (CR) rates of Groups 1, 2, 3 and 4 were 74.5% (41/55), 45.9% (19/41), 17.5% (7/40) and 38.8% (19/49), respectively. The CR rate was higher in Group 1 than in the other 3 groups (34.6%, 45/130) (P = 0.000). A significant correlation was found between CR rate and the number of chemotherapeutic courses (P = 0.023). The main adverse reactions included bone marrow suppression and secondary infection. Conclusion  FA regimen is a good choice for patients with AML, especially those who have failed to achieve CR after the first course of induction chemotherapy. Supported by a grant from the Planned Science and Technology Project of Guangzhou (No. 2006Z3-E0401).     

Polymeric nanoparticle-mediated silencing of CD44 receptor in CD34 acute myeloid leukemia cells

The adhesion receptor CD44 plays an important role in the survival and retention of leukemic stem/progenitor cells (LSPC) within the bone marrow (BM) niche, as well as in the high relapse rates of acute myeloid leukemia (AML). Down-regulating CD44 could be clinically relevant not only for suppression of the deregulated function of LSPC but also in LSPC response to chemotherapeutic agents. Small interfering RNA (siRNA) delivery is a promising approach for AML treatment, and we recently reported effective siRNA delivery into difficult-to-transfect AML cell lines using lipid-substituted polyethylenimine/siRNA complexes (polymeric nanoparticles). In this study, we investigated polymeric nanoparticle-mediated silencing of CD44 in CD34⁺ LSPC cell models (leukemic KG-1 and KG-1a cell lines) as well as primary AML cells. Polymeric nanoparticle-mediated silencing decreased surface CD44 levels in KG-1, KG-1a and primary AML cells by up to 27%, 30% and 20% at day 3, respectively. Moreover, CD44 silencing resulted in induction of apoptosis in KG-1 cells, reduced adhesion of KG-1 and KG-1a cells to hyaluronic acid-coated cell culture plates and BM-MSC, and decreased adhesion of primary AML cells to BM-MSC. Our results suggest that polymeric nanoparticle-mediated silencing of CD44 might be a useful technique for inhibiting LSPC interactions with their microenvironment, thereby prohibiting leukemia progression or sensitizing LSPC to chemotherapy.     

Clinical and proteomic characterization of acute myeloid leukemia with mutated RAS

BACKGROUND:

Activating mutations in RAS are frequently present in patients with acute myeloid leukemia (AML), but their overall prognostic impact is not clear.

METHODS:

A retrospective analysis was performed to establish the clinical characteristics of patients with RAS‐mutated (RAS^mut) AML, to analyze their outcome by therapy, and to describe the proteomic profile of RAS^mut compared with wild‐type RAS (RAS^WT) AML.

RESULTS:

Of 609 patients with newly diagnosed AML, 11% had RAS^mut. Compared with RAS^WT, patients with RAS^mut AML were younger (median age, 54 years vs 63 years; P = .001), had a higher white blood cell count (16K mm^?3 vs 4K mm^?3 ; P < 0.001) and bone marrow blast percentage (56% vs 42%; P = .01) at diagnosis, and were less likely to have an antecedent hematologic disorder (36% vs 50%; P = .03). The inv(16) karyotype was overrepresented in patients with RAS^mut and the ?5 and/or ?7 karyotype was underrepresented. RAS mutations were found to have no prognostic impact on overall survival or disease‐free survival overall or within cytogenetic subgroups. There was a suggestion that patients with RAS^mut benefited from cytarabine (AraC)‐based therapy. Proteomic analysis revealed simultaneous upregulation of the RAS‐Raf‐MAP kinase and phosphoinositide 3‐kinase (PI3K) signaling pathways in patients with RAS^mut.

CONCLUSIONS:

RAS mutations in AML may delineate a subset of patients who benefit from AraC‐based therapy and who may be amenable to treatment with inhibitors of RAS and PI3K signaling pathways. Cancer 2012. © 2012 American Cancer Society.     

表达PD-1 shRNA增强靶向CD19 CAR-T细胞对肿瘤细胞的杀伤能力

目的：设计和构建表达PD-1 shRNA的靶向CD19 CAR-T细胞并验证其体外肿瘤细胞杀伤能力。方法：设计并构建 表达PD-1 shRNA的CD19 CAR分子基因,将其包装成逆转录病毒载体,通过qPCR法检测病毒载体拷贝数。将慢病毒转导人原 代T细胞,获得三种CAR-T细胞,分别为RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞。 采用qPCR法检测三种CAR-T细胞中PD-1 mRNA的表达水平,流式细胞术检测三种CAR-T细胞中PD-1表达水平,萤光素酶报 告基因实验、流式细胞术检测在不同效靶比时 CAR-T 细胞对 CD19 阳性靶细胞（人淋巴瘤 daudi 细胞）的杀伤功能。结果： RNAU6-CD19 CAR、PD-1 shRNA1-CD19 CAR、PD-1 shRNA2-CD19 CAR 三种 CAR 分子成功包装成逆转录病毒载体,病毒载 体拷贝数均高于 1×107 拷贝/mL,转导人原代 T 细胞获得 CAR-T 细胞,RNAU6-CD19 CAR-T、PD-1 shRNA1-CD19 CAR-T、PD-1 shRNA2-CD19 CAR-T细胞转导效率分别为43.1%、55.1%、41.7%。与RNAU6-CD19 CAR-T细胞相比,PD-1 shRNA1-CD19 CAR-T、 PD-1 shRNA2-CD19 CAR-T细胞中PD-1 mRNA表达水平均显著降低（均P<0.01）、细胞表面PD-1表达水平更低（均P<0.01）、体 外对daudi细胞的杀伤率更高（P<0.05或P<0.01）。结论：成功构建表达PD-1 shRNA的靶向CD19 CAR-T细胞,其对CD19阳性 靶细胞的杀伤率显著提高,PD-1 mRNA及其翻译产物PD-1的表达减少,CAR-T细胞的耗竭减缓。