急性髓细胞性白血病细胞表面CD分子表达及临床意义研究

目的 探讨急性髓细胞性白血病(AML)细胞表面CD分子的表达情况.方法 采用CD45分子作为靶目标抗原,应用多参数流式细胞术检测300例AML患者细胞表面的CD分子,分析患者的免疫表型.结果以表达百分比﹥20%作为阳性,300例AML患者的CD抗原表达均不相同,cyt-MPO、CD33、CD117和CD38表达最强,为强阳性(80%~100%).以CD抗原表达>60%为阳性,结果显示:CD33、CD117、CD13、cyt-MPO四种抗原阳性表达的患者,阳性细胞所占的比例高于其他抗原,差异有统计学意义(P﹤0.05).CD15/CD64、CD15/CD33和CD33/CD64抗原的阳性表达比较,差异均有统计学意义(P﹤0.05);CD33/cyt-MPO抗原的阳性表达比较,差异无统计学意义(P﹥0.05).结论 AML细胞表面的CD抗原表达具有差异性,CD13、cyt-MPO、CD33、CD117呈高表达.CD分子在AML细胞中阳性表达的差异性可为治疗提供有价值的线索.     

急性白血病CD11a、CD54的表达及其临床意义

目的:观察与探讨CD11a、CD54在急性白血病中的表达变化及其临床意义。方法:采用流式细胞术检测了20例急性淋巴细胞白血病(acutelymphoblasticleukemia,ALL),27例急性髓性白血病(autemyeloidleukemia,AML)及16例正常对照骨髓单个核细胞CD11a和CD54的表达率。结果:ALL组及AML组CD11a、CD54表达率均显著低于对照组(P<0.01);化疗后ALL及AML完全缓解者(CR)CD11a、CD54表达与对照组无差异(P>0.05),非CR者仍显著低于对照组(P<0.01);ALL及AML髓外浸润组CD11a表达率显著高于无浸润组(P<0.01)。结论:CD11a、CD54在急性白血病中表达异常,可能作为观察疗效、提示预后的指标之一。     

抗B-CLL-IgM Id×抗CD3双功能抗体对LAK细胞杀伤活性的影响

抗肿瘤双特异性抗体(bispecific antibody,BsAb)由于具有同时与效应细胞及靶瘤细胞两种不同抗原特异性结合的双重特异性,从而提高了效应细胞对靶瘤细胞的特异性杀伤活性,因此,其在肿瘤靶向生物治疗中的应用日益受到重视.本研究通过体外实验,观察了抗人慢性B淋巴细胞白血病LgM独特型×抗CD3双特异性抗体(抗B-CLL-LgM Id×抗CD3 BsAb)对LAK细胞杀伤靶瘤细胞活性的影响,现报道如下.     

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。     

微型双功能抗体介导人T细胞杀伤耐药实体瘤细胞

目的 探讨抗P-糖蛋白（P-gP）／抗CD3微型双功能抗体介导人T细胞杀伤耐药实体瘤细胞的作用。方法 采用抗E-tag亲和层析柱分离纯化抗P-gp／抗CD3微型双功能抗体,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）进行鉴定;采用^51Cr释放实验,测定抗P-gP／抗CD3微型双功能抗体介导的人T细胞体外靶向杀伤活性;采用多药耐药（MDR）细胞系KB／MDR、敏感KB细胞裸鼠移植瘤模型,测定该微型双功能抗体介导的体内靶向杀伤活性。结果 纯化的抗P-gp／抗CD3微型双功能抗体,在相对分子质量28000和26000处各有1条蛋白带。在抗P-gp／抗CD3微型双功能抗体存在的情况下,激活的T淋巴细胞能够裂解KB／MDR细胞,且随着效靶比的增高而增高。抗P-gp／抗CD3微型双功能抗体联合人T淋巴细胞能有效抑制KB／MDR细胞裸鼠移植瘤的生长,而对敏感KB细胞移植瘤的生长无抑制作用。结论 抗P-gP／抗CD3微型双功能抗体在体内外均能介导人T淋巴细胞杀伤表达P-gP抗原的KB／MDR细胞,是有望用于耐药实体瘤临床治疗的双特异性抗体。     

CD20抗原及治疗性抗CD20抗体

CD20是人类B淋巴细胞表面特有的标识.它高表达于所有正常B细胞和多数恶性B细胞表面,不会发生明显的内化和脱落,是治疗非霍奇金淋巴瘤(non-Hodgkin's lymphoma,NHL)理想的靶抗原.其胞外区由43个氨基酸残基组成,但组成的抗原表位却异常多样.目前,已经有多种针对CD20抗原的抗体被FDA批准上市,用于B细胞非霍奇金淋巴瘤的治疗,都显示出良好的效果.     

CD20 和CD19 双靶点CAR-T细胞对B淋巴细胞肿瘤的杀伤作用

目的：设计同时靶向B淋巴细胞表面CD20 和CD19 两个抗原蛋白的双特异嵌合抗原受体（chimeric antigen receptor,CAR）并制备BiCAR-T 细胞,检测其对B淋巴细胞肿瘤细胞的杀伤作用以及对免疫缺陷B-NSG 小鼠白血病模型的治疗效果。方法：构建基于鼠源CD19 和人源化CD20 scFv 的双靶点CAR分子,将CAR基因装载于慢病毒载体中,在293T细胞中包装慢病毒,感染健康人T 细胞制备BiCAR-T 细胞。构建表达CD19 和CD20 的K562-CD19-GFP、K562-CD20-GFP 以及表达Luciferase 的Nalm6-Luc-GFP 细胞作为靶细胞,将BiCAR-T 细胞与靶细胞共同孵育,检测其对靶细胞杀伤能力及释放IFN-γ 水平。使用Nalm6-Luc-GFP 细胞构建白血病小鼠模型,尾静脉注射BiCAR-T细胞,通过小动物成像方法观察BiCAR-T细胞对荷瘤小鼠的治疗作用。结果：健康人来源的BiCAR-T 细胞经7 d 培养后扩增良好,扩增倍数为20~50 倍,阳性率为10%~92%,显示成功制备BiCAR-T细胞。在效靶比为10∶1 时,BiCAR-T细胞对Nalm-6、K562-CD19-GFP 和K562-CD20-GFP 的杀伤率显著高于对照组细胞[ （76.7±7.4）% vs（8.7±2.4）%、（93.3±5.2）% vs（46.7±6.2）、（51.0±0.8）vs（30.7±0.5）%,均P<0.01];与对照组相比,与Nalm-6细胞共孵育后BiCAR-T 细胞分泌IFN- γ 量显著增加[（872.7±7.7）vs（101.0±5.3）pg/ml ,P<0.01]。动物实验表明,BiCAR-T细胞对B-NSG小鼠白血病模型治疗效果明显,注射BiCAR-T细胞后白血病细胞逐渐减少,第50 天时基本消失,小鼠全部存活至第70 天被安乐死;PBS和对照T细胞组小鼠分别在（19±3）和（20±1）d 全部死亡。结论：成功设计了表达CD19 和CD20 的双靶点CAR分子后成功制备了BiCAR-T 细胞,该细胞能够有效杀伤表达CD19 和/或CD20 的B淋巴细胞肿瘤细胞,与靶细胞共同孵育后能够分泌大量IFN-γ,对B-NSG免疫缺陷小鼠白血病模型具有明显的治疗效果。     

CD13、CD33在BCR-ABL+急性淋巴细胞白血病患者骨髓中的表达及其与临床特征和预后的相关性

目的:探讨髓系抗原CD13、CD33在BCR-ABL+急性淋巴细胞白血病（acute lymphoblastic leukemia,ALL）患者骨髓中的表达及其与临床特征和预后的相关性。方法:应用实时荧光定量聚合酶链反应（real time quantitative-polymerase chain reaction,RQ-PCR）检测119例初诊ALL患者骨髓BCR-ABL表达水平。用流式细胞学检查检测ALL的抗原表达水平。对照分析单纯BCR-ABL+患者中CD13+和CD13-以及CD33+和CD33-患者在初次诱导缓解率（initial-induction remission rate,CR1）和总生存率（overall survival rate,OS）的区别。结果:在BCR-ABL+ 的ALL患者中,初诊时CD13+患者较CD13-患者具有较低的首次诱导缓解率,差异具统计学意义（P＜0.05）,但CD33+患者较CD33-患者首次诱导缓解率无统计学差异。CD13+组及CD33+的OS显著低于CD13-组及CD33-组（P＜0.05）。结论:BCR-ABL+ALL患者中CD13+及CD33+提示预后不良。     

双功能抗体抗CD3/抗CD19介导T细胞对靶细胞的杀伤作用

背景与目的:双功能抗体有2个抗原结合臂,一方面具有肿瘤相关抗原,另一方面具有和免疫效应细胞表面分子标记结合的能力,并能有效地使效应细胞靶向杀火肿瘤细胞的作用.本实验探讨抗CD3/抗CD19双功能抗体介导T细胞杀伤靶细胞的作用.方法:利用非放射性荧光染料Calcein-AM进行抗体介导的体外特异性杀伤活性榆测,Ficoll-Hypaque法分离外周血淋巴细胞(peripheral blood lymphocyte,PBL),流式细胞术从中分选T淋巴细胞及CD4+T淋巴细胞和CD8+T淋巴细胞作为效应细胞.首先以Raji细胞为靶细胞,按不同效靶比,不同抗体浓度分组,以PBS、抗CD3scFv+、抗CD3scFv抗体及非相关抗体抗CD3/抗PGP为对照,另设CD19-K562细胞为非相关靶细胞组,比较双功能抗体介导特异性体外杀伤活性.取上述杀伤实验效靶比25:1,抗体浓度500 ng/mL各组,ELISA法比较激活的T细胞分泌IL-2的量,Real-time PcR法检测T细胞因子IL-3、IFN-γ、TNF-α激活后的表达变化.结果:体外介导T细胞杀伤靶细胞Raji的效果比对照组明显增强(P＜0.05),激活T细胞分泌IL-2、IL-3、IFN-γ及TNF-α的表达均明显高于对照组.结论:双功能抗体抗CD3/抗CD19在体外介导T细胞对靶细胞具有较强的杀伤作用.     

急性白血病细胞中CD19的表达

目的观察CD19在急性白血病(AL)中的表达与分布,为白血病的诊断、鉴别以及导向治疗提供依据.方法采用27个荧光直标单克隆抗体(单抗)及CD45/SSC双参数设门多色流式细胞术对321例AL细胞进行免疫诊断和分型,并对CD19在各类型AL细胞中的表达情况进行分析.结果在116例B细胞系急性淋巴细胞性白血病(ALL)患者中,CD19的阳性率(99.1%)明显高于B细胞系其它相关性抗原的阳性率;CD19在17例含B细胞系成分的杂合型白血病(HAL)中全部表达,而在29例T细胞系ALL和7例T/My HAL则均无表达;在152例急性髓系白血病(AML)中,仅11例(7.2%)CD19阳性,明显低于其在B细胞系ALL中的阳性率(P=0.001);CD22在B/My HAL的阳性率(12/15,80.0%)明显高于CD19 -AML(0/11,0%,P＜0.001).结论 CD19对B系ALL细胞的特异性较好,敏感性较高,是诊断B细胞系ALL较为可靠的表面标记,也可作为导向治疗B细胞系ALL的理想靶点.     

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。      

Reduced effect of gemtuzumab ozogamicin (CMA-676) on P-glycoprotein and/or CD34-positive leukemia cells and its restoration by multidrug resistance modifiers.

Gemtuzumab ozogamicin (CMA-676), a calicheamicin-conjugated humanized anti-CD33 mouse monoclonal antibody, has recently been introduced clinically as a promising drug for the treatment of patients with acute myeloid leukemia (AML), more than 90% of which express CD33 antigen. However, our recent study suggested that CMA-676 was excreted by a multi- drug-resistance (MDR) mechanism in P-glycoprotein (P-gp)-expressing leukemia cell lines. We analyzed the in vitro effects of CMA-676 on leukemia cells from 27 AML patients in relation to the amount of P-gp, MDR-associated protein 1 (MRP1), CD33 and CD34, using a multi-laser-equipped flow cytometer. The cytocidal effect of CMA-676, estimated by the amount of hypodiploid portion on cell cycle, was inversely related to the amount of P-gp estimated by MRK16 monoclonal antibody (P = 0.004), and to the P-gp function assessed by intracellular rhodamine-123 accumulation in the presence of PSC833 or MS209 as a MDR modifier (P = 0.0004 and P = 0.002, respectively). In addition, these MDR modifiers reversed CMA-676 resistance in P-gp-expressing CD33(+) leukemia cells (P = 0.001 with PSC833 and P = 0.0007 with MS209). In CD33(+) AML cells from 13 patients, CMA-676 was less effective on CD33(+)CD34(+) than CD33(+)CD34(-) cells (P = 0.002). PSC833 partially restored the effect of CMA-676 in CD33(+)CD34(+) cells. These results suggest that the combined use of CMA-676 and a MDR modifier will be more effective on CD33(+) AML with P-gp-related MDR.     

GM-CSF does not increase CD20 antigen expression on chronic lymphocytic leukemia lymphocytes

CD20 antigen expression in B-chronic lymphocytic leukemia (B-CLL) is at significantly lower levels than in non-Hodgkins lymphoma, which may affect the degree of anti-CD20 antibody binding. Low density of CD20 expression on malignant cells may explain the lower response rates to anti-CD20 monoclonal antibody, observed in B-CLL. Upregulating the antigen receptor intensity on tumor cells may enhance the response rates. In this study, we examined the influence of granulocyte macrophage-colony stimulating factor (GM-CSF) on the expression level of CD20 antigen and percent of cells expressing CD20 antigen on B-CLL lymphocytes, in vivo. CD20 antigen expression was studied by flow cytometry at baseline, 12 and 24 h after GM-CSF injection. However neither upregulation of CD20 antigen nor a change of the proportion of CD20 positive cells was observed after a dose of 5 microg/kg GM-CSF. Strategies other than GM-CSF priming needs to be evaluated in order to increase the efficacy of anti-CD20 monoclonal antibodies in B-CLL.     

Prognostic impact of CD45 antigen expression in high-risk, childhood B-cell precursor acute lymphoblastic leukemia.

To evaluate the clinical implications of CD45 expression in acute childhood lymphoblastic leukemia (ALL), we measured the CD45 expression of blast cells from 133 untreated patients with childhood B-precursor ALL (n = 118) or T-ALL (n = 15). CD45 expression (> or = 20%) was detected in all 15 cases (100%) of T-ALL, and 101 cases (86%) of B-precursor ALL. In 122 cases, the fluorescence intensity of the CD45 expression was measured as a relative value; the ratio of average linear values (RALV) of CD45 on the blasts to that on CD3-positive T-lymphocytes from the same specimen. The expression was more intense in the T-ALL cases than in the B-precursor ALL cases (RALV, mean +/- SE: T-ALL 0.230 +/- 0.04 vs. pro-B ALL 0.150 +/- 0.012/pre-B ALL 0.153 +/- 0.019, p < 0.05). However, the intensity of the CD10, CD19, CD20 and CD34 antigen immunoreactivity did not correlate with the CD45 expression. Patients with hyperdiploidy (chromosome number > 50) showed significantly lower levels of CD45 expression than patients with t(1;19) or normal karyotypes (RALV, mean +/- SE: 0.081 +/- 0.022 vs. 0.133 +/- 0.03/0.143 +/- 0.019, p < 0.05). Other clinical features such as age, gender and WBC count did not correlate with CD45 expression. The prognostic implications of CD45 expression were studied in non-high-risk (low-risk + intermediate-risk) (n = 60) and high-risk patients (n = 52) with B-precursor ALL who had been treated with the risk-directed protocol of ALL-941 trial. Although CD45 expression did not correlate with the event-free survival (EFS) of the non-high-risk patients, there was a significant correlation between the expression levels and the EFS of the high-risk patients: the 3-year EFS rate of the CD45low group (n = 26, RALV = 0.017-0.132) was 88 +/- 7% versus the CD45high group (n = 26, RALV = 0.133-0.450) at 34 +/- 24% (p < 0.05). These results show that the levels of expression of the CD45 antigen on leukemic lymphoblasts are significantly correlated with the clinical features and prognosis of childhood ALL.     

Frequent expression of MDR1 and MDR3 genes in acute myelocytic leukemia cells with t(8;21) (q22;q22)

Multidrug resistance (MDR) 1 and MDR3 gene expression were examined in acute myelocytic leukemia (AML) cells from 126 Japanese patients. In 119 AML patients at diagnosis 52 were revealed to have P-gp/MDR1 (43.7%). AML cases with t(8;21) had a higher incidence of P-gp expression (13/17; 76.5%) than cases with other karyotypes (33/89; 37.1%) (p=0.0062). CD19(+) cases expressed P-gp frequently (17/25) as did CD7(+) cases (24/35). In 17 CD19(+) AML with P-gp expression, 12 had t(8;21) abnormality. In 68 AML samples examined, MDR3 mRNA was detected in 11 cases, 9 of which had the t(8;21) abnormality. The MDR3(+) t(8;21) AML samples were also positive for CD19. We analyzed P-gp expression at both diagnosis and relapse in 18 AML patients. All 11 P-gp(+) cases at relapse, in which 5 patients were P-gp negative at diagnosis, showed either t(8;21) or CD7 positivity. Our data demonstrated that expression of MDR1 and MDR3 in AML is closely associated with chromosome abnormality t(8;21) and expression of immature lymphoid antigen CD19 as well as CD7. Kasumi-1, a t(8;21) AML cell line, was demonstrated to lose P-gp by the treatment of 1,25(OH)(2)D-3, a monocyte/macropharge differentiation inducer, suggesting the possible contribution to the therapy for AML.     

白血病bcl-2,p170,CD34表达的临床意义及其相关性

目的：探讨和分析白血病ｂｃｌ－２,ｐ１７９０,ＣＤ３４表达的临床意义及相相关性。方法：Ｅ和ＡＢＣ免疫细胞化学法检测急性白血病和２３例慢性粒细胞白血病ｂｃｌ－２,Ｐ１７０,ＣＤ３４４的表达。结果：复发、难治急性白血病ｂｃｌ－２、ｐ１７０、ＣＤ３４的表达率明显高于初治急性白血病;ｂｃｌ－２,Ｐ１７０,ＣＤ３４高表达的急性白血病完全缓解率显著低于低于不表达或低表达者;加速／急变期的慢性粒细胞白血病（ＣＤ     

儿童急性髓系白血病免疫表型特点分析

目的:分析儿童急性髓系白血病(AML)免疫表型特征,探讨其临床意义.方法:采用四色流式细胞术CD45/SSC双参数散点图设门方法,对127例儿童AML患者幼稚细胞进行免疫表型检测,对抗原表达情况进行分析.结果:在127例儿童急性髓系白血病患者中,髓系特异性抗原CD33、CD13和CD117的表达最常见,分别达95.3%、90.6%、90.6%.造血干/祖细胞抗原CD34、HLA-DR、CD38阳性率分别达53.5%、71.6%和97.6%.有65.4%的病例伴有淋系抗原的表达,其中以CD56的表达最常见占38.6%,其次为CD7占21.3%.有淋系抗原表达(LymAg+)患者早期抗原CD34和HLA-DR抗原表达阳性率明显高于无淋系抗原表达(LymAg-)患者(P<0.05).结论:免疫分型对儿童AML的诊断和不同亚型鉴别具有重要意义.     

CD45设门多参数流式细胞术在急性白血病免疫表型分析中的应用

目的：分析急性白血病的抗原表达及其临床意义。方法：采用一组系列相关单抗直接免疫荧光标记CD45设门的多参数流式细胞术,检测35例急性白血病患者的免疫表型。结果：11例ALL中B－ALL8例,T－ALL3例,其中出现髓系抗原表达4例,占36．4％,CD34表达10例,占90．1％;24例AML中伴淋系抗原表达7例,占29．17％,CD34表达16例,占70．8％,DR的表达与CD34一致,5例M3患者均无CD34和HLA DR表达。伴髓系统原表达的ALL CR率低于髓系抗原阴性表达者（1／3及5／6）,但统计学上差异无显著性（P＞0．05）;伴淋系抗原表达的AML患者CR率明显低于淋系抗原阴性表达者（0／5及10／10）,两组间差异具有显著性（P＜0．01）。结论：CD45设门的多参数流式细胞术是分析白血病免疫表型的最好方法,白血病抗原的错义表达是预后不良的因素之一。