2E8-NCTD-脂质体体外靶向B系白血病干细胞及机制研究
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摘要
研究背景和目的:
B系淋巴细胞白血病是儿童常见的恶性肿瘤,目前化疗仍是对其治疗的主要手段。虽然大部分患儿对初始化疗反应较好,但约有20%左右的患儿由于不能耐受常规化疗非特异性毒副作用,导致化疗失败或白血病复发。最近研究发现:从B淋巴细胞白血病患儿的体内分选出CD34+CD19+CD38+/-的细胞注射入新生的NOD/SCID小鼠体内能长出B系白血病细胞,而CD34+CD19-CD38-CD10-的细胞注射入新生的NOD/SCID小鼠体内则表现为正常的细胞表型。可见CD34+CD19+CD38+/-细胞具有自我更新和无限增殖的能力,其也完全不同于髓系白血病干细胞(AML-LSC),是一类新的干细胞群——B系LSCs。因此,CD19是B系LSCs及其子代白血病细胞表面的特异性标记,是靶向杀伤B系LSC及其子代白血病细胞的良好靶点。
目前以单克隆抗体(单抗)为基础的靶向治疗是白血病治疗最有前途的方法之一。CD20和CD33等单抗已在临床分别广泛应用于B系非霍杰金淋巴瘤(B-NHL)和AML的治疗。免疫脂质体是靶向治疗的分支之一,具有单抗导向和脂质体药物传输的双重特性。将免疫脂质体包封抗肿瘤药物,不但可将大量的药物分子特异地传输到肿瘤细胞,还可显著减少对正常组织的非特异性毒性作用。据报道大量的药物分子甚至可以克服肿瘤耐药。目前,免疫脂质体治疗B系血液肿瘤的研究已取的了令人鼓舞的结果,如CD19靶向的柔红霉素(DXR)和长春新碱(VCR)脂质体等在体外能显著增加对CD19+的白血病细胞的细胞毒作用,在体内能显著延长B系模型小鼠的生存时间,CD19靶向的伊马替尼(imatinib)脂质体能将大量的伊马替尼分子传输到SUP-15细胞内,克服了低剂量imatinib所导致的imatinib耐药问题。然而目前报道的免疫脂质体不能有效清除LSCs,因为象DXR和VCR等常规化疗药不能作用于静止期的LSCs。
去甲斑蝥素(Norcantharidin,NCTD)是干细胞特异性的杀伤药物,其通过抑制干细胞相关的抗凋亡轴——即下调(HLF/SLUG)的活性,诱导AML-LSCs凋亡。但NCTD对正常HSCs的杀伤作用在一定程度上削弱了其对LSCs的治疗优势。
ZCH-4-2E8(简称2E8)是本实验室自行研制的抗人CD19单抗的新克隆,在先前的研究中,我们已证实2E8—NCTD免疫毒素对CD19+的白血病细胞具有明显的靶向杀伤作用。
本研究拟采用免疫脂质体技术,将NCTD包封于脂质体中,并与2E8单抗连接,研制成2E8-NCTD-liposomes,观察该免疫脂质体靶向识别并杀伤B系LSCs及其子代白血病细胞的作用,阐明其靶向杀伤的机制,为其进行体内试验奠定基础。
方法:
(1) 2E8单克隆抗体的制备:采用凝胶过滤层析法从腹水中纯化2E8单抗;流式细胞术检测其对CD19高表达的Nalm-6细胞的靶向效率;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)鉴定抗体纯度及分子量。
(2) 2E8单克隆抗体修饰的空白脂质体(blank-liposomes)的制备及评价:采用分光光度计建立Stewart分析法的标准曲线(检测脂质体中磷脂的含量)。采用后插技术将2E8与blank-liposomes连接,制备2E8-blank-liposomes;并用流式细胞术证明两者是否连接成功(为做流式检测,Rhodamine标记的磷脂酰乙醇胺——PE,加入脂质体的磷脂中);结合Stewart分析法和BCA蛋白浓度检测法考察2E8和Mal-PEG_(2000)-DSPE摩尔比对脂质体表面抗体密度的影响;SDS-PAGIE验证2E8是否以完整的形式插入脂质体表面;激光粒度测定仪检测其粒径并观察其在不同溶液中放置不同时间是否稳定。流式细胞术评价2E8-blank-liposomes和blank-liposomes对不同细胞株(Nalm-6,Raji,Molt-3,K562)和健康者外周血中T、B淋巴细胞的靶向效率以及靶向结合的稳定性;流式细胞术检测游离2E8阻滞2E8-blank-liposomes对CD19抗原的特异性结合能力;激光共聚焦显微镜和流式细胞术(木瓜酶法)观察2E8-blank-liposomes在Nalm-6细胞中的内化作用。
(3) 2E8单抗修饰的去甲斑蝥素脂质体(NCTD-liposomes)的制备及评价
首先建立脂质体包封率检测的高效液相色谱法(HPLC)。采用薄膜分散法制备NCTD-liposomes,并通过后插技术将2E8与NCTD-liposomes连接,制备2E8-NCTD-liposomes。HPLC测定脂质体的包封率;激光粒度测定仪检测其粒径;流式细胞术和激光共聚焦显微镜评价2E8-NCTD-liposomes和NCTD-liposomes对不同细胞株(Nalm-6,Raji,Molt-3,K562)的靶向效率和差异;流式细胞术检测B淋巴细胞白血病病人骨髓及Nalm-6细胞株中B系LSC(CD34+CD19+CD38+/-)的靶向效率;MTT法评价2E8-NCTD-liposomes,NCTD-liposomes和游离的NCTD对不同细胞株(Nalm-6,Molt-3)的细胞毒作用,并计算各自IC50值。
(4) 2E8-NCTD-liposomes的靶向杀伤机制
采用激光共聚焦显微镜和HPLC观察2E8-NCTD-liposomes通过CD19受体介导的内化作用将NCTD转运至细胞内的现象。HE染色和琼脂糖凝胶电泳观察2E8-NCTD-liposomes处理后Nalm-6细胞发生凋亡改变(凋亡小体和DNA ladder);流式细胞术(Annexin V-FITC和PI双标法)检测2E8-NCTD-liposomes处理后Nalm-6细胞的早期凋亡,并定量检测相同浓度的2E8-NCTD-liposomes,NCTD-liposomes和游离的NCTD对Nalm-6和Molt-3凋亡的影响以及不同浓度的2E8-NCTD-liposomes对Nalm-6细胞凋亡的影响。实时荧光定量PCR的方法检测2E8-NCTD-liposomes处理后Nalm-6细胞中与B系LSCs相关的抗凋亡因子HLF和其下游的促凋亡因子NFIL 3在转录水平的相对表达规律。
结果:
(1)2E8单克隆抗体的制备及鉴定:从Balb/C小鼠腹水中纯化获得高纯度2E8抗体蛋白,经流式细胞术检测,2E8对Nalm-6细胞的靶向识别效率为99.93%,接近于标准进口单抗(阳性对照)CD19 PE对Nalm-6细胞的靶向识别效率(99.94%)。SDS-PAGE鉴定2E8的重链和轻链的分子量分别约为70 kDa和23kDa。
(2) 2E8单抗修饰的空白脂质体(blank-liposomes)的制备及评价:随着2E8/Mal-PEG_(2000)-DSPE的摩尔比增加,平均每个脂质体表面抗体的密度逐渐增加,但抗体与脂质体的连接效率逐渐下降。当2E8/Mal-PEG_(2000)-DSPE的摩尔比为1:50,Mal-PEG_(2000)-DSPE与2E8的连接位点达到饱和。流式细胞术结果显示:当2E8-blank-liposomes与Nalm-6细胞孵育后,在右上象限可观察到Rhodamine标记的脂质体的红色荧光(直标法)与GAM-FITC标记2E8的绿色荧光(间标法)成一一对应的直线关系,而在T淋巴细胞白血病细胞株Molt-3细胞中未观察到相似的结果,提示2E8与脂质体连接成功。粒径测定仪检测发现:2E8-blank-liposomes和非靶向的blank-liposomes平均粒径分别为121.25±4.32纳米(nm)和106.32±3.21纳米(nm);并将其分别放置于HBS缓冲液,含3%吐温80的HBS缓冲液(v/v)中和含50%小牛血清的HBS缓冲液(v/v)中,4℃,至24天,其粒径无显著变化。SDS-PAGE证实2E8-blank-liposomes在制备过程中每一步2E8单抗均以完整抗体的形式插入脂质体表面。2E8-blank-liposomes对Nalm-6和Raji细胞靶向效率(直标法和间标法)几乎接近于100%,显著高于其对Molt-3和急性红白血病细胞株K562细胞的靶向效率(P＜0.01),也显著高于空白非靶向脂质体(blank-liposomes)对Nalm-6和Raji细胞的靶向识别效率(P＜0.01);且随着孵育时间的延长和孵育介质的改变(PBS,50%小牛血清,50%人血浆),2E8-blank-liposomes与Nalm-6细胞的结合稳定,平均荧光强度无显著变化。游离的2E8能阻滞2E8-blank-liposomes与Nalm-6细胞的特异性结合。2E8-blank-liposomes对外周血中正常B淋巴细胞的靶向效率达(97.65±1.30)%,显著高于对正常T细胞的靶向效率((4.70±1.78)%,P＜0.01)。激光共聚焦显微镜和流式细胞术观察到2E8-blank-liposomes在30分钟至8小时内被内化入Nalm-6细胞。
(3) 2E8-NCTD-liposomes的制备及评价:采用HPLC测定脂质体的包封率为46.51%,粒径测定仪发现2E8-NCTD-liposomes和NCTD-liposomes的平均粒径分别是118.32nm和104.12nm;与2E8-blank-liposomes相似,2E8-NCTD-liposomes对Nalm-6和Raji细胞的靶向识别效率(直标法和间标法)显著高于其对Molt-3和K562细胞的靶向识别效率(P＜0.01),也显著高于非靶向blank-liposomes对Nalm-6和Raji细胞靶向效率(P＜0.01);2ES-NCTD-liposomes对B淋巴细胞白血病患者骨髓中CD34+CD19+CD38+和CD34+CD19+CD38-细胞的靶向效率分别为98.00%和92.45%(间标法),NCTD-liposomes对CD34+CD19+CD38+和CD34+CD19+CD38-细胞的靶向效率不到5%,两者差异具有显著性(P＜0.01);激光共聚焦显微镜观察发现在Nalm-6细胞表面同时存在Rhodamine的红色和GAM-FITC的绿色荧光信号,而在Molt-3细胞表面则未观察到相似的结果。MTT法检查显示在24-72小时和10-50μM浓度范围内,2E8-NCTD-liposomes对Nalm-6细胞的杀伤呈时间和剂量的依赖关系。相同浓度的NCTD处理细胞24-72小时后,2E8-NCTD-liposomes对Nalm-6细胞的抑制率高于NCTD-liposomes和游离NCTD对Nalm-6细胞的抑制率;也显著高于2E8-NCTD-liposomes对Molt-3细胞的抑制率(P＜0.01);2E8-NCTD-liposomes对Nalm-6细胞的IC50值低于NCTD-liposomes和游离NCTD对Nalm-6细胞的IC50值;也显著低于2E8-NCTD-liposomes对Molt-3细胞的IC50值(P＜0.01)。
(4)2E8-NCTD-liposomes的靶向杀伤机制:激光共聚焦显微镜和HPLC的结果证实:2E8-NCTD-liposomes通过CD19受体介导内化入Nalm-6细胞中。前者观察到Nalm-6细胞在胞浆内出现逐渐累积的红色荧光(Rhodamine);后者显示:在Nalm-6细胞中2E8-NCTD-liposomes对NCTD的转运效率是在Molt-3细胞中的4倍多;也是NCTD-liposomes对NCTD的转运效率的2倍多。IC50浓度的2E8-NCTD-liposomes处理后的Nalm-6细胞,HE染色的结果显示其出现细胞皱缩,核固缩破碎,凋亡小体形成等典型的凋亡改变;琼脂糖凝胶电泳显示其出现典型的晚期凋亡的DNA ladder条带。流式细胞术的结果也显示:2E8-NCTD-liposomes诱导Nalm-6细胞凋亡呈剂量依赖关系,在相同浓度(IC50浓度/2E8-NCTD-liposomes)下处理细胞72小时,2E8-NCTD-liposomes对Nalm-6细胞的凋亡比例((47.55±0.02)%)显著高于其对Molt-3细胞的凋亡比例((8.65±0.01)%,P＜0.01);也高于NCTD-liposomes((27.09±0.01)%)或游离的NCTD((24.22±0.02)%)对Nalm-6细胞的凋亡比例(P＜0.05)。实时荧光定量PCR的结果显示:IC50浓度的2E8-NCTD-liposomes处理Nalm-6细胞48小时后HLF基因相对表达(0.62±0.09)较未处理前(1)降低(P＜0.01),NFIL3基因相对表达(1.47±0.02)较未处理前(1)增加(P＜0.01)。
结论:
1、成功研制CD19抗体修饰的免疫脂质体2E8-blank-liposomes和2E8-NCTD-liposomes;
2、2E8-blank-liposomes可作为靶向B系LSC(CD34CD19+CD38+/-)及其子代白血病细胞(CD19+)有用的药物载体;
3、2E8-NCTD-liposomes可对B系白血病细胞产生靶向细胞杀伤作用;
4、2E8-NCTD-liposomes靶向杀伤B系白血病细胞的作用机制是通过CD19受体介导而发生内化,NCTD被转运至细胞内释放,诱导靶细胞凋亡;
5、2E8-NCTD-liposomes诱导B系LSC凋亡与抑制LSC相关的抗凋亡轴——HLF/SLUG轴的活性密切相关,即在转录水平与下调LSC相关的抗凋亡因子HLF的表达和上调HLF下游的促凋亡因子NFIL3的表达密切相关。
Background and aims
B lineage lymphocytic leukemia(B-lineage ALL) is a common hematopoietic malignancy in children.Chemotherapy is currently the principal treatment modality of the disease.Despite the treatment outcome has recently been improved significantly with this type of therapy,about 20%of the patients remain treatment failure due to either the systemic nonspecific cytotoxicities caused by the therapeutic agents or the disease relapse with incomplete eradication of all the leukemia cells.A recent study has demonstrated that when purified CD34+ CD38 + CD19+ and CD34+CD38-CD19+ bone marrow(BM) or peripheral blood(PB) cells from pediatric B-lineage ALL patients were intravenously injected into lethally irradiated newborn non obese diabetes/severe combined immunodeficiency(NOD/SCID/IL2rc) null mice, CD34+CD38+CD19+ and CD34+CD38-CD19+ cells were able to initiate B-lineage ALL in primary recipients,whereas purified CD34+CD38-CD19-CD10- cells showed normal human hematopoietic repopulation.Furthermore,transplantation of CD34+CD38+CD19+ cells resulted in the development of B lineage ALL in secondary recipients.Clearly,the CD34+CD19+CD38+/CD38- cell populations possess the capacity for indefinite proliferation and self-renewal which are currently named a new class of leukemia stem cells(LSC),B-lineage LSCs.Therefore,the CD19 antigen is a specific marker of B lineage ALL which can be an excellent target to both B-lineage LSCs and their progeny.
Monoclonal antibody(mAb)-based targeted therapy is one of the most promising methods for leukemia.CD20 and CD33 monoclonal antibody have been widely used for the treatment of B lineage non-Hodgkin's lymphoma(B-NHL) and of acute myeloid leukemia(AML),respectively.The immunoliposome is one branch of targeting therapies with double features of monoclonal antibody-oriented and liposome drug delivery.Immunoliposomes encapsulating anticancer drugs not only can specifically transfer a large number of drug molecules to tumor cells for tumor kill,but also significantly reduce the non-specific cytotoxicity of the drugs to normal tissues.It has been reported that immunoliposomes can even overcome the resistance of anticancer drugs.Currently,the encouraging results via immunoliposomes have been used in studying the treatments of hematologic malignancies.For instances,anti-CD 19 targeted liposomal anticancer drugs,i.e.doxorubicin(DOX) or vincristine(VCR) have been seen the association with increased specific cyotoxicity to B lineage leukemia cells relative to non-targeted liposomes in vitro,they can also prolong the survival in mice compared to non-targeted liposomes or free DXR in vivo.Anti-CD19 targeted liposomal imatinib can transfer a large number of imatinib molecules to SUP-15 cells,so it overcomes the resistance of the leukemia cells to imatinib which are not inhibited by lower concentration of the drug.However,these reported immunoliposomes can not effectively kill the B-lineage LSCs because the chemotherapeutic agents encapsulated in the liposomes,such as DOX,VCR and imatinib are unable to exert the cytotoxicity to LSCs in quiescent state.
Norcantharidin(NCTD) is a stem cell-specific agent capable of inducing apoptosis in AML-LSCs by inhibiting the stem cell-related anti-apoptotic axis - the HLF/SLUG activity.But,NCTD killing effect on normal HSCs nonspecifically attenuated the advantages of its treatment to LSCs.
ZCH-4-2E8(2E8) is a novel anti-human CD19 monoclonal antibody generated in our laboratory.In previous studies,we have confirmed that 2E8-NCTD immunotoxin confers a clear role in targeting killing of CD19+ leukemic cells.
In this study,we intended to develop 2E8-NCTD-liposomes by encapsulating NCTD into liposomes modified with the 2E8 monoclonal antibody;to observe the targeting recognition and killing of B lineage LSCs and their progeny;to elucidate the targeting killing mechanism of 2E8-NCTD-liposomes to build the foundation for further development of the therapeutic agent 2E8-NCTD-liposomes in vivo.
Methods
(1) Preparation of 2E8 monoclonal antibody and identification:The ascites containing 2E8 antibody(Ab) derived from Balb/c mice were purified by gel filtration chromatography-SephacryL S-300-HR column.The targeting efficiency of 2E8 on CD19+ Nalm-6 cells was detected by flow cytometry(FCM).The purity and molecular weight(MW) of the heavy and light chains were identified by SDS-PAGE.
(2) Preparation and evaluation of 2E8-blank-liposomes:A standard curve of Stewart analysis was established using spectrophotometer(detection of the content of phospholipid in liposomes);2E8 antibody was connected with blank-liposomes by using post-incorporation technology;the successful link between 2E8 antibody and blank-liposomes was proved by using FCM.The effects of 2E8 and Mal-PEG_(2000)-DSPE lipid molar ratio of the antibody density on the surface of liposomes were determined by using Stewart analysis and BCA protein quantification assay Kit.The mean particle size and stability of 2E8-blank-liposomes were determined by a laser particle size analyzer.The targeting efficiency of 2E8-blank-liposomes to different cell lines and normal T,B lymphocytes and the binding stability were evaluated by using FCM.The internalization of 2E8-blank-liposomes in Nalm-6 cells was observed by using FCM and Laser scanning confocal microscope.
(3) Preparation and evaluation of 2E8-NCTD-liposomes:NCTD-liposomes were prepared by the method of thin-film dispersion.The rate of liposome encapsulation was quantified by high performance liquid chromatography(HPLC).The mean particle size of 2E8-NCTD-liposomes was determined by the laser particle size analyzer.The targeting efficiency of 2E8-NCTD-liposomes to different cell lines and B lineage LSCs was evaluated by using FCM.The targeting discrepancy of 2E8-NCTD-liposomes to different cell lines was observed by Laser scanning confocal microscope.The cytotoxicity of 2E8-NCTD-liposomes,NCTD- liposomes and free NCTD to different cell lines were assayed by MTT method.
(4) The targeting killing mechanism of 2E8-NCTD-liposomes:The phenomenon of CD19 receptor-mediated internalization of 2E8-NCTD-liposomes was demonstrated by Laser scanning confocal microscope and HPLC.The apoptosis changes of Nalm-6 cells treated by 2E8-NCTD-liposomes were observed through HE staining and agarose gel electrophoresis.The early apoptosis of Nalm-6 cells treated by 2E8-NCTD-liposomes,NCTD-liposomes and free NCTD were quantitatively assessed by FCM.The relative expressions of anti-apoptosis factors-HLF related to B lineage LSCs and its downstream pro-apoptotic factor- NFIL 3 in Nalm-6 treated by 2E8-NCTD-liposomes at the transcriptional level was detected by real-time fluorescence quantitative PCR method.
Results
(1) Preparation and identification of 2E8 monoclonal antibody:The targeting efficiency of 2E8 antibody on CD19+ Nalm-6 was 99.93%,close to that(99.94%) of a positive control antibody CD19 PE.The heavy chain and light chain molecular weight of 2E8 were approximately 70 kDa and 23 kDa,respectively.
(2) Preparation and evaluation of 2E8-blank-liposomes:As the molar ratio of 2E8/Mal-PEG2000-DSPE increase,the antibody density on the surface of liposomes gradually increased,but the coupling efficiency of antibodies gradually decreased. When the 2E8/Mal-PEG2000-DSPE molar ratio was 1:50,the site of Mal-PEG2000-DSPE connection with the 2E8 was saturated.2E8 antibody was successfully linked to liposomes with the evidence of one to one linear relationship between liposomes labeled with Rhodamine(red fluorescence) and 2E8 antibody labeled with GAM-FITC(green fluorescence) in the upper right quadrant in Nalm-6 cells co-cultured with 2E8-blank-liposomes.The average particle size of 2E8-blank-liposomes and blank-liposomes were 121.25 nm and 106.30nm, respectively;and after they were placed in the HBS buffer(containing 3%of Tween 80 or the HBS buffer containing 50%calf serum in 4℃) up to 24 days,no significant changes in their average sizes were observed.During the course of preparing 2E8-blank-liposomes,the 2E8 antibody molecules were incorporated into the surface of liposomes in their intact conformation.The targeting efficiencies of 2E8-blank-liposomes on the Nalm-6 or Raji cells in either direct or indirect method were almost close to 100%,significantly higher than that on Molt-3 or acute erythroleukemia cell line K562 cells(P＜0.01).It was also significantly higher than that of non-targeted liposomes on Nalm-6 or Raji cells(P＜0.01).And with the extension of the incubation time and changes in incubation medium(PBS,50%fetal calf serum,50%human plasma),the binding of 2E8-blank-liposomes to Nalm-6 cells was stable with no significant changes in the average fluorescence intensity. Free 2E8 can block the specific binding of 2E8-blank-liposomes to Nalm-6 cells. The targeting efficiency of 2E8-blank-liposomes on the peripheral blood of normal B lymphocytes was(97.65±1.30)%,significantly higher than that on normal T cells ((4.70±1.78)%,P＜0.01).2E8-blank-liposomes can be internalized into Nalm-6 cells in the incubation periods of 30mins-8hs.
(3) Preparation and evaluation of 2ES-NCTD-liposomes:The encapsulating efficiency of NCTD-liposomes was 46.51%.The average particle size of 2E8-NCTD-liposomes and NCTD-liposomes were 118.32 nm and 104.12 nm, respectively;Similar to 2E8-blank-liposomes,the targeting efficiency of 2E8-NCTD-liposomes on the Nalm-6 or Raji cells significantly higher than that on Molt-3 or K562 cells(P＜0.01).It was also significantly higher than that of non-targeted liposomes on Nalm-6 or Raji cells(P＜0.01).The targeting efficiency of 2E8-NCTD-liposomes on CD34+CD19+CD38+ or CD34+CD19+CD38-cells were 98.00%or 92.45%,while that of NCTD-liposomes on both the cell subpopulations were less than 5%(P＞0.05).Red Rhodamine and green GAM-FITC fluorescence signals co-existed on the surface of Nalm-6 cells,whereas similar results were not observed on the surface of Molt-3 cells.The killing effects of 2E8-NCTD-liposomes on the Nalm-6 cells for 24-72 hrs showed a time- and dose-dependent manner in 10-50μM range of concentration.When the Nalm-6 cells were treated with free NCTD and 2E8-NCTD-liposomes for 24-72 hrs,the inhibitory rate of Nalm-6 cells by 2E8-NCTD-liposomes was significantly higher than that by the same concentration of NCTD-liposomes or free NCTD;It was also significantly higher than that of Molt-3 cells inhibited by the 2E8-NCTD liposomes (P＜0.01);The IC50 value of 2E8-NCTD-liposomes was significantly lower that that of NCTD-liposomes and free NCTD on Nalm-6 cells;It also was significantly lower than that of 2E8-NCTD-liposomes on Molt-3 cells.
(4) The targeting killing mechanism of 2E8-NCTD-liposomes: 2E8-NCTD-liposomes were internalized into Nalm-6 cells through the CD19 receptor with the evidence of gradual accumulation of red fluorescence in the cytoplasm of Nalm-6 cells.The transferring efficiency of 2E8-NCTD-lipsomes for NCTD in Nalm-6 cells was four times more than that in Molt-3 cells;It was also two times more than that of NCTD-liposomes for NCTD in Nalm-6 cells.There were typical apoptotic changes,including cell shrinkage,nuclear condensation and fragmentation,apoptotic body formation,in Nalm-6 cells treated by the IC50 concentration of 2E8-NCTD-liposomes.Agarose gel electrophoresis of 2E8-NCTD-liposome-treated Naml-6 cells revealed a ladder-like pattern of DNA fragments,indicating the apoptosis induction was involved in the process of cell kill. 2E8-NCTD-liposomes induce the apoptosis of Nalm-6 cells in a dose-dependent manner.The apoptotic percentage of Nalm-6 cells((47.55±0.02)%) induced by the IC50 concentration of 2Eg-NCTD-liposomes was significantly higher than that induced by the same concentrations of either free NCTD((24.22±0.02)%,P＜0.05) %or NCTD-liposomes((27.09±0.01)%,P＜0.05)%for the same period of incubation time;It was also significantly higher than that of Molt-3 cells induced by the same concentration of 2E8-NCTD-liposomes.The relative expression of HLF decreased while that of NFIL 3 increased in Nalm-6 treated by the IC50 concentration of 2E8-NCTD-liposomes for 48 hrs in mRNA level,compared to that in untreated Nalm-6 cells.
Conclusions:
(1) CD19 mAb modified immunoliposomes,i.e.2E8-blank-liposomes and 2E8-NCTD-liposomes are successfully prepared.
(2) 2E8-blank-liposomes can be used as a useful drug carrier targeting B lineage LSC (CD34CD19 + CD38 +/-) and their progeny.
(3) 2E8-NCTD-liposomes can exert targeted cytotoxicity to CD19+ leukemia cells.
(4) The targeting killing mechanism of 2E8-NCTD-liposomes is that NCTD is transferred into CD19+ leukemia cells through CD19 mediating internalization of 2E8-NCTD-liposomes and induced apoptosis of the targeted cells.
(5) 2ES-NCTD-liposomes-induced apoptosis of B lineage LSCs is closely associated with inhibiting the LSC-related anti-apoptotic axis,namely,reducing the expression of the LSC-related anti-apoptotic factor-HLF gene and increasing the expression of the downstream pro-apoptotic factor-NFIL3 gene.
B系淋巴细胞白血病是儿童常见的恶性肿瘤,目前化疗仍是对其治疗的主要手段。虽然大部分患儿对初始化疗反应较好,但约有20%左右的患儿由于不能耐受常规化疗非特异性毒副作用,导致化疗失败或白血病复发。最近研究发现:从B淋巴细胞白血病患儿的体内分选出CD34+CD19+CD38+/-的细胞注射入新生的NOD/SCID小鼠体内能长出B系白血病细胞,而CD34+CD19-CD38-CD10-的细胞注射入新生的NOD/SCID小鼠体内则表现为正常的细胞表型。可见CD34+CD19+CD38+/-细胞具有自我更新和无限增殖的能力,其也完全不同于髓系白血病干细胞(AML-LSC),是一类新的干细胞群——B系LSCs。因此,CD19是B系LSCs及其子代白血病细胞表面的特异性标记,是靶向杀伤B系LSC及其子代白血病细胞的良好靶点。
目前以单克隆抗体(单抗)为基础的靶向治疗是白血病治疗最有前途的方法之一。CD20和CD33等单抗已在临床分别广泛应用于B系非霍杰金淋巴瘤(B-NHL)和AML的治疗。免疫脂质体是靶向治疗的分支之一,具有单抗导向和脂质体药物传输的双重特性。将免疫脂质体包封抗肿瘤药物,不但可将大量的药物分子特异地传输到肿瘤细胞,还可显著减少对正常组织的非特异性毒性作用。据报道大量的药物分子甚至可以克服肿瘤耐药。目前,免疫脂质体治疗B系血液肿瘤的研究已取的了令人鼓舞的结果,如CD19靶向的柔红霉素(DXR)和长春新碱(VCR)脂质体等在体外能显著增加对CD19+的白血病细胞的细胞毒作用,在体内能显著延长B系模型小鼠的生存时间,CD19靶向的伊马替尼(imatinib)脂质体能将大量的伊马替尼分子传输到SUP-15细胞内,克服了低剂量imatinib所导致的imatinib耐药问题。然而目前报道的免疫脂质体不能有效清除LSCs,因为象DXR和VCR等常规化疗药不能作用于静止期的LSCs。
去甲斑蝥素(Norcantharidin,NCTD)是干细胞特异性的杀伤药物,其通过抑制干细胞相关的抗凋亡轴——即下调(HLF/SLUG)的活性,诱导AML-LSCs凋亡。但NCTD对正常HSCs的杀伤作用在一定程度上削弱了其对LSCs的治疗优势。
ZCH-4-2E8(简称2E8)是本实验室自行研制的抗人CD19单抗的新克隆,在先前的研究中,我们已证实2E8—NCTD免疫毒素对CD19+的白血病细胞具有明显的靶向杀伤作用。
本研究拟采用免疫脂质体技术,将NCTD包封于脂质体中,并与2E8单抗连接,研制成2E8-NCTD-liposomes,观察该免疫脂质体靶向识别并杀伤B系LSCs及其子代白血病细胞的作用,阐明其靶向杀伤的机制,为其进行体内试验奠定基础。
方法:
(1) 2E8单克隆抗体的制备:采用凝胶过滤层析法从腹水中纯化2E8单抗;流式细胞术检测其对CD19高表达的Nalm-6细胞的靶向效率;十二烷基磺酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)鉴定抗体纯度及分子量。
(2) 2E8单克隆抗体修饰的空白脂质体(blank-liposomes)的制备及评价:采用分光光度计建立Stewart分析法的标准曲线(检测脂质体中磷脂的含量)。采用后插技术将2E8与blank-liposomes连接,制备2E8-blank-liposomes;并用流式细胞术证明两者是否连接成功(为做流式检测,Rhodamine标记的磷脂酰乙醇胺——PE,加入脂质体的磷脂中);结合Stewart分析法和BCA蛋白浓度检测法考察2E8和Mal-PEG_(2000)-DSPE摩尔比对脂质体表面抗体密度的影响;SDS-PAGIE验证2E8是否以完整的形式插入脂质体表面;激光粒度测定仪检测其粒径并观察其在不同溶液中放置不同时间是否稳定。流式细胞术评价2E8-blank-liposomes和blank-liposomes对不同细胞株(Nalm-6,Raji,Molt-3,K562)和健康者外周血中T、B淋巴细胞的靶向效率以及靶向结合的稳定性;流式细胞术检测游离2E8阻滞2E8-blank-liposomes对CD19抗原的特异性结合能力;激光共聚焦显微镜和流式细胞术(木瓜酶法)观察2E8-blank-liposomes在Nalm-6细胞中的内化作用。
(3) 2E8单抗修饰的去甲斑蝥素脂质体(NCTD-liposomes)的制备及评价
首先建立脂质体包封率检测的高效液相色谱法(HPLC)。采用薄膜分散法制备NCTD-liposomes,并通过后插技术将2E8与NCTD-liposomes连接,制备2E8-NCTD-liposomes。HPLC测定脂质体的包封率;激光粒度测定仪检测其粒径;流式细胞术和激光共聚焦显微镜评价2E8-NCTD-liposomes和NCTD-liposomes对不同细胞株(Nalm-6,Raji,Molt-3,K562)的靶向效率和差异;流式细胞术检测B淋巴细胞白血病病人骨髓及Nalm-6细胞株中B系LSC(CD34+CD19+CD38+/-)的靶向效率;MTT法评价2E8-NCTD-liposomes,NCTD-liposomes和游离的NCTD对不同细胞株(Nalm-6,Molt-3)的细胞毒作用,并计算各自IC50值。
(4) 2E8-NCTD-liposomes的靶向杀伤机制
采用激光共聚焦显微镜和HPLC观察2E8-NCTD-liposomes通过CD19受体介导的内化作用将NCTD转运至细胞内的现象。HE染色和琼脂糖凝胶电泳观察2E8-NCTD-liposomes处理后Nalm-6细胞发生凋亡改变(凋亡小体和DNA ladder);流式细胞术(Annexin V-FITC和PI双标法)检测2E8-NCTD-liposomes处理后Nalm-6细胞的早期凋亡,并定量检测相同浓度的2E8-NCTD-liposomes,NCTD-liposomes和游离的NCTD对Nalm-6和Molt-3凋亡的影响以及不同浓度的2E8-NCTD-liposomes对Nalm-6细胞凋亡的影响。实时荧光定量PCR的方法检测2E8-NCTD-liposomes处理后Nalm-6细胞中与B系LSCs相关的抗凋亡因子HLF和其下游的促凋亡因子NFIL 3在转录水平的相对表达规律。
结果:
(1)2E8单克隆抗体的制备及鉴定:从Balb/C小鼠腹水中纯化获得高纯度2E8抗体蛋白,经流式细胞术检测,2E8对Nalm-6细胞的靶向识别效率为99.93%,接近于标准进口单抗(阳性对照)CD19 PE对Nalm-6细胞的靶向识别效率(99.94%)。SDS-PAGE鉴定2E8的重链和轻链的分子量分别约为70 kDa和23kDa。
(2) 2E8单抗修饰的空白脂质体(blank-liposomes)的制备及评价:随着2E8/Mal-PEG_(2000)-DSPE的摩尔比增加,平均每个脂质体表面抗体的密度逐渐增加,但抗体与脂质体的连接效率逐渐下降。当2E8/Mal-PEG_(2000)-DSPE的摩尔比为1:50,Mal-PEG_(2000)-DSPE与2E8的连接位点达到饱和。流式细胞术结果显示:当2E8-blank-liposomes与Nalm-6细胞孵育后,在右上象限可观察到Rhodamine标记的脂质体的红色荧光(直标法)与GAM-FITC标记2E8的绿色荧光(间标法)成一一对应的直线关系,而在T淋巴细胞白血病细胞株Molt-3细胞中未观察到相似的结果,提示2E8与脂质体连接成功。粒径测定仪检测发现:2E8-blank-liposomes和非靶向的blank-liposomes平均粒径分别为121.25±4.32纳米(nm)和106.32±3.21纳米(nm);并将其分别放置于HBS缓冲液,含3%吐温80的HBS缓冲液(v/v)中和含50%小牛血清的HBS缓冲液(v/v)中,4℃,至24天,其粒径无显著变化。SDS-PAGE证实2E8-blank-liposomes在制备过程中每一步2E8单抗均以完整抗体的形式插入脂质体表面。2E8-blank-liposomes对Nalm-6和Raji细胞靶向效率(直标法和间标法)几乎接近于100%,显著高于其对Molt-3和急性红白血病细胞株K562细胞的靶向效率(P＜0.01),也显著高于空白非靶向脂质体(blank-liposomes)对Nalm-6和Raji细胞的靶向识别效率(P＜0.01);且随着孵育时间的延长和孵育介质的改变(PBS,50%小牛血清,50%人血浆),2E8-blank-liposomes与Nalm-6细胞的结合稳定,平均荧光强度无显著变化。游离的2E8能阻滞2E8-blank-liposomes与Nalm-6细胞的特异性结合。2E8-blank-liposomes对外周血中正常B淋巴细胞的靶向效率达(97.65±1.30)%,显著高于对正常T细胞的靶向效率((4.70±1.78)%,P＜0.01)。激光共聚焦显微镜和流式细胞术观察到2E8-blank-liposomes在30分钟至8小时内被内化入Nalm-6细胞。
(3) 2E8-NCTD-liposomes的制备及评价:采用HPLC测定脂质体的包封率为46.51%,粒径测定仪发现2E8-NCTD-liposomes和NCTD-liposomes的平均粒径分别是118.32nm和104.12nm;与2E8-blank-liposomes相似,2E8-NCTD-liposomes对Nalm-6和Raji细胞的靶向识别效率(直标法和间标法)显著高于其对Molt-3和K562细胞的靶向识别效率(P＜0.01),也显著高于非靶向blank-liposomes对Nalm-6和Raji细胞靶向效率(P＜0.01);2ES-NCTD-liposomes对B淋巴细胞白血病患者骨髓中CD34+CD19+CD38+和CD34+CD19+CD38-细胞的靶向效率分别为98.00%和92.45%(间标法),NCTD-liposomes对CD34+CD19+CD38+和CD34+CD19+CD38-细胞的靶向效率不到5%,两者差异具有显著性(P＜0.01);激光共聚焦显微镜观察发现在Nalm-6细胞表面同时存在Rhodamine的红色和GAM-FITC的绿色荧光信号,而在Molt-3细胞表面则未观察到相似的结果。MTT法检查显示在24-72小时和10-50μM浓度范围内,2E8-NCTD-liposomes对Nalm-6细胞的杀伤呈时间和剂量的依赖关系。相同浓度的NCTD处理细胞24-72小时后,2E8-NCTD-liposomes对Nalm-6细胞的抑制率高于NCTD-liposomes和游离NCTD对Nalm-6细胞的抑制率;也显著高于2E8-NCTD-liposomes对Molt-3细胞的抑制率(P＜0.01);2E8-NCTD-liposomes对Nalm-6细胞的IC50值低于NCTD-liposomes和游离NCTD对Nalm-6细胞的IC50值;也显著低于2E8-NCTD-liposomes对Molt-3细胞的IC50值(P＜0.01)。
(4)2E8-NCTD-liposomes的靶向杀伤机制:激光共聚焦显微镜和HPLC的结果证实:2E8-NCTD-liposomes通过CD19受体介导内化入Nalm-6细胞中。前者观察到Nalm-6细胞在胞浆内出现逐渐累积的红色荧光(Rhodamine);后者显示:在Nalm-6细胞中2E8-NCTD-liposomes对NCTD的转运效率是在Molt-3细胞中的4倍多;也是NCTD-liposomes对NCTD的转运效率的2倍多。IC50浓度的2E8-NCTD-liposomes处理后的Nalm-6细胞,HE染色的结果显示其出现细胞皱缩,核固缩破碎,凋亡小体形成等典型的凋亡改变;琼脂糖凝胶电泳显示其出现典型的晚期凋亡的DNA ladder条带。流式细胞术的结果也显示:2E8-NCTD-liposomes诱导Nalm-6细胞凋亡呈剂量依赖关系,在相同浓度(IC50浓度/2E8-NCTD-liposomes)下处理细胞72小时,2E8-NCTD-liposomes对Nalm-6细胞的凋亡比例((47.55±0.02)%)显著高于其对Molt-3细胞的凋亡比例((8.65±0.01)%,P＜0.01);也高于NCTD-liposomes((27.09±0.01)%)或游离的NCTD((24.22±0.02)%)对Nalm-6细胞的凋亡比例(P＜0.05)。实时荧光定量PCR的结果显示:IC50浓度的2E8-NCTD-liposomes处理Nalm-6细胞48小时后HLF基因相对表达(0.62±0.09)较未处理前(1)降低(P＜0.01),NFIL3基因相对表达(1.47±0.02)较未处理前(1)增加(P＜0.01)。
结论:
1、成功研制CD19抗体修饰的免疫脂质体2E8-blank-liposomes和2E8-NCTD-liposomes;
2、2E8-blank-liposomes可作为靶向B系LSC(CD34CD19+CD38+/-)及其子代白血病细胞(CD19+)有用的药物载体;
3、2E8-NCTD-liposomes可对B系白血病细胞产生靶向细胞杀伤作用;
4、2E8-NCTD-liposomes靶向杀伤B系白血病细胞的作用机制是通过CD19受体介导而发生内化,NCTD被转运至细胞内释放,诱导靶细胞凋亡;
5、2E8-NCTD-liposomes诱导B系LSC凋亡与抑制LSC相关的抗凋亡轴——HLF/SLUG轴的活性密切相关,即在转录水平与下调LSC相关的抗凋亡因子HLF的表达和上调HLF下游的促凋亡因子NFIL3的表达密切相关。
Background and aims
B lineage lymphocytic leukemia(B-lineage ALL) is a common hematopoietic malignancy in children.Chemotherapy is currently the principal treatment modality of the disease.Despite the treatment outcome has recently been improved significantly with this type of therapy,about 20%of the patients remain treatment failure due to either the systemic nonspecific cytotoxicities caused by the therapeutic agents or the disease relapse with incomplete eradication of all the leukemia cells.A recent study has demonstrated that when purified CD34+ CD38 + CD19+ and CD34+CD38-CD19+ bone marrow(BM) or peripheral blood(PB) cells from pediatric B-lineage ALL patients were intravenously injected into lethally irradiated newborn non obese diabetes/severe combined immunodeficiency(NOD/SCID/IL2rc) null mice, CD34+CD38+CD19+ and CD34+CD38-CD19+ cells were able to initiate B-lineage ALL in primary recipients,whereas purified CD34+CD38-CD19-CD10- cells showed normal human hematopoietic repopulation.Furthermore,transplantation of CD34+CD38+CD19+ cells resulted in the development of B lineage ALL in secondary recipients.Clearly,the CD34+CD19+CD38+/CD38- cell populations possess the capacity for indefinite proliferation and self-renewal which are currently named a new class of leukemia stem cells(LSC),B-lineage LSCs.Therefore,the CD19 antigen is a specific marker of B lineage ALL which can be an excellent target to both B-lineage LSCs and their progeny.
Monoclonal antibody(mAb)-based targeted therapy is one of the most promising methods for leukemia.CD20 and CD33 monoclonal antibody have been widely used for the treatment of B lineage non-Hodgkin's lymphoma(B-NHL) and of acute myeloid leukemia(AML),respectively.The immunoliposome is one branch of targeting therapies with double features of monoclonal antibody-oriented and liposome drug delivery.Immunoliposomes encapsulating anticancer drugs not only can specifically transfer a large number of drug molecules to tumor cells for tumor kill,but also significantly reduce the non-specific cytotoxicity of the drugs to normal tissues.It has been reported that immunoliposomes can even overcome the resistance of anticancer drugs.Currently,the encouraging results via immunoliposomes have been used in studying the treatments of hematologic malignancies.For instances,anti-CD 19 targeted liposomal anticancer drugs,i.e.doxorubicin(DOX) or vincristine(VCR) have been seen the association with increased specific cyotoxicity to B lineage leukemia cells relative to non-targeted liposomes in vitro,they can also prolong the survival in mice compared to non-targeted liposomes or free DXR in vivo.Anti-CD19 targeted liposomal imatinib can transfer a large number of imatinib molecules to SUP-15 cells,so it overcomes the resistance of the leukemia cells to imatinib which are not inhibited by lower concentration of the drug.However,these reported immunoliposomes can not effectively kill the B-lineage LSCs because the chemotherapeutic agents encapsulated in the liposomes,such as DOX,VCR and imatinib are unable to exert the cytotoxicity to LSCs in quiescent state.
Norcantharidin(NCTD) is a stem cell-specific agent capable of inducing apoptosis in AML-LSCs by inhibiting the stem cell-related anti-apoptotic axis - the HLF/SLUG activity.But,NCTD killing effect on normal HSCs nonspecifically attenuated the advantages of its treatment to LSCs.
ZCH-4-2E8(2E8) is a novel anti-human CD19 monoclonal antibody generated in our laboratory.In previous studies,we have confirmed that 2E8-NCTD immunotoxin confers a clear role in targeting killing of CD19+ leukemic cells.
In this study,we intended to develop 2E8-NCTD-liposomes by encapsulating NCTD into liposomes modified with the 2E8 monoclonal antibody;to observe the targeting recognition and killing of B lineage LSCs and their progeny;to elucidate the targeting killing mechanism of 2E8-NCTD-liposomes to build the foundation for further development of the therapeutic agent 2E8-NCTD-liposomes in vivo.
Methods
(1) Preparation of 2E8 monoclonal antibody and identification:The ascites containing 2E8 antibody(Ab) derived from Balb/c mice were purified by gel filtration chromatography-SephacryL S-300-HR column.The targeting efficiency of 2E8 on CD19+ Nalm-6 cells was detected by flow cytometry(FCM).The purity and molecular weight(MW) of the heavy and light chains were identified by SDS-PAGE.
(2) Preparation and evaluation of 2E8-blank-liposomes:A standard curve of Stewart analysis was established using spectrophotometer(detection of the content of phospholipid in liposomes);2E8 antibody was connected with blank-liposomes by using post-incorporation technology;the successful link between 2E8 antibody and blank-liposomes was proved by using FCM.The effects of 2E8 and Mal-PEG_(2000)-DSPE lipid molar ratio of the antibody density on the surface of liposomes were determined by using Stewart analysis and BCA protein quantification assay Kit.The mean particle size and stability of 2E8-blank-liposomes were determined by a laser particle size analyzer.The targeting efficiency of 2E8-blank-liposomes to different cell lines and normal T,B lymphocytes and the binding stability were evaluated by using FCM.The internalization of 2E8-blank-liposomes in Nalm-6 cells was observed by using FCM and Laser scanning confocal microscope.
(3) Preparation and evaluation of 2E8-NCTD-liposomes:NCTD-liposomes were prepared by the method of thin-film dispersion.The rate of liposome encapsulation was quantified by high performance liquid chromatography(HPLC).The mean particle size of 2E8-NCTD-liposomes was determined by the laser particle size analyzer.The targeting efficiency of 2E8-NCTD-liposomes to different cell lines and B lineage LSCs was evaluated by using FCM.The targeting discrepancy of 2E8-NCTD-liposomes to different cell lines was observed by Laser scanning confocal microscope.The cytotoxicity of 2E8-NCTD-liposomes,NCTD- liposomes and free NCTD to different cell lines were assayed by MTT method.
(4) The targeting killing mechanism of 2E8-NCTD-liposomes:The phenomenon of CD19 receptor-mediated internalization of 2E8-NCTD-liposomes was demonstrated by Laser scanning confocal microscope and HPLC.The apoptosis changes of Nalm-6 cells treated by 2E8-NCTD-liposomes were observed through HE staining and agarose gel electrophoresis.The early apoptosis of Nalm-6 cells treated by 2E8-NCTD-liposomes,NCTD-liposomes and free NCTD were quantitatively assessed by FCM.The relative expressions of anti-apoptosis factors-HLF related to B lineage LSCs and its downstream pro-apoptotic factor- NFIL 3 in Nalm-6 treated by 2E8-NCTD-liposomes at the transcriptional level was detected by real-time fluorescence quantitative PCR method.
Results
(1) Preparation and identification of 2E8 monoclonal antibody:The targeting efficiency of 2E8 antibody on CD19+ Nalm-6 was 99.93%,close to that(99.94%) of a positive control antibody CD19 PE.The heavy chain and light chain molecular weight of 2E8 were approximately 70 kDa and 23 kDa,respectively.
(2) Preparation and evaluation of 2E8-blank-liposomes:As the molar ratio of 2E8/Mal-PEG2000-DSPE increase,the antibody density on the surface of liposomes gradually increased,but the coupling efficiency of antibodies gradually decreased. When the 2E8/Mal-PEG2000-DSPE molar ratio was 1:50,the site of Mal-PEG2000-DSPE connection with the 2E8 was saturated.2E8 antibody was successfully linked to liposomes with the evidence of one to one linear relationship between liposomes labeled with Rhodamine(red fluorescence) and 2E8 antibody labeled with GAM-FITC(green fluorescence) in the upper right quadrant in Nalm-6 cells co-cultured with 2E8-blank-liposomes.The average particle size of 2E8-blank-liposomes and blank-liposomes were 121.25 nm and 106.30nm, respectively;and after they were placed in the HBS buffer(containing 3%of Tween 80 or the HBS buffer containing 50%calf serum in 4℃) up to 24 days,no significant changes in their average sizes were observed.During the course of preparing 2E8-blank-liposomes,the 2E8 antibody molecules were incorporated into the surface of liposomes in their intact conformation.The targeting efficiencies of 2E8-blank-liposomes on the Nalm-6 or Raji cells in either direct or indirect method were almost close to 100%,significantly higher than that on Molt-3 or acute erythroleukemia cell line K562 cells(P＜0.01).It was also significantly higher than that of non-targeted liposomes on Nalm-6 or Raji cells(P＜0.01).And with the extension of the incubation time and changes in incubation medium(PBS,50%fetal calf serum,50%human plasma),the binding of 2E8-blank-liposomes to Nalm-6 cells was stable with no significant changes in the average fluorescence intensity. Free 2E8 can block the specific binding of 2E8-blank-liposomes to Nalm-6 cells. The targeting efficiency of 2E8-blank-liposomes on the peripheral blood of normal B lymphocytes was(97.65±1.30)%,significantly higher than that on normal T cells ((4.70±1.78)%,P＜0.01).2E8-blank-liposomes can be internalized into Nalm-6 cells in the incubation periods of 30mins-8hs.
(3) Preparation and evaluation of 2ES-NCTD-liposomes:The encapsulating efficiency of NCTD-liposomes was 46.51%.The average particle size of 2E8-NCTD-liposomes and NCTD-liposomes were 118.32 nm and 104.12 nm, respectively;Similar to 2E8-blank-liposomes,the targeting efficiency of 2E8-NCTD-liposomes on the Nalm-6 or Raji cells significantly higher than that on Molt-3 or K562 cells(P＜0.01).It was also significantly higher than that of non-targeted liposomes on Nalm-6 or Raji cells(P＜0.01).The targeting efficiency of 2E8-NCTD-liposomes on CD34+CD19+CD38+ or CD34+CD19+CD38-cells were 98.00%or 92.45%,while that of NCTD-liposomes on both the cell subpopulations were less than 5%(P＞0.05).Red Rhodamine and green GAM-FITC fluorescence signals co-existed on the surface of Nalm-6 cells,whereas similar results were not observed on the surface of Molt-3 cells.The killing effects of 2E8-NCTD-liposomes on the Nalm-6 cells for 24-72 hrs showed a time- and dose-dependent manner in 10-50μM range of concentration.When the Nalm-6 cells were treated with free NCTD and 2E8-NCTD-liposomes for 24-72 hrs,the inhibitory rate of Nalm-6 cells by 2E8-NCTD-liposomes was significantly higher than that by the same concentration of NCTD-liposomes or free NCTD;It was also significantly higher than that of Molt-3 cells inhibited by the 2E8-NCTD liposomes (P＜0.01);The IC50 value of 2E8-NCTD-liposomes was significantly lower that that of NCTD-liposomes and free NCTD on Nalm-6 cells;It also was significantly lower than that of 2E8-NCTD-liposomes on Molt-3 cells.
(4) The targeting killing mechanism of 2E8-NCTD-liposomes: 2E8-NCTD-liposomes were internalized into Nalm-6 cells through the CD19 receptor with the evidence of gradual accumulation of red fluorescence in the cytoplasm of Nalm-6 cells.The transferring efficiency of 2E8-NCTD-lipsomes for NCTD in Nalm-6 cells was four times more than that in Molt-3 cells;It was also two times more than that of NCTD-liposomes for NCTD in Nalm-6 cells.There were typical apoptotic changes,including cell shrinkage,nuclear condensation and fragmentation,apoptotic body formation,in Nalm-6 cells treated by the IC50 concentration of 2E8-NCTD-liposomes.Agarose gel electrophoresis of 2E8-NCTD-liposome-treated Naml-6 cells revealed a ladder-like pattern of DNA fragments,indicating the apoptosis induction was involved in the process of cell kill. 2E8-NCTD-liposomes induce the apoptosis of Nalm-6 cells in a dose-dependent manner.The apoptotic percentage of Nalm-6 cells((47.55±0.02)%) induced by the IC50 concentration of 2Eg-NCTD-liposomes was significantly higher than that induced by the same concentrations of either free NCTD((24.22±0.02)%,P＜0.05) %or NCTD-liposomes((27.09±0.01)%,P＜0.05)%for the same period of incubation time;It was also significantly higher than that of Molt-3 cells induced by the same concentration of 2E8-NCTD-liposomes.The relative expression of HLF decreased while that of NFIL 3 increased in Nalm-6 treated by the IC50 concentration of 2E8-NCTD-liposomes for 48 hrs in mRNA level,compared to that in untreated Nalm-6 cells.
Conclusions:
(1) CD19 mAb modified immunoliposomes,i.e.2E8-blank-liposomes and 2E8-NCTD-liposomes are successfully prepared.
(2) 2E8-blank-liposomes can be used as a useful drug carrier targeting B lineage LSC (CD34CD19 + CD38 +/-) and their progeny.
(3) 2E8-NCTD-liposomes can exert targeted cytotoxicity to CD19+ leukemia cells.
(4) The targeting killing mechanism of 2E8-NCTD-liposomes is that NCTD is transferred into CD19+ leukemia cells through CD19 mediating internalization of 2E8-NCTD-liposomes and induced apoptosis of the targeted cells.
(5) 2ES-NCTD-liposomes-induced apoptosis of B lineage LSCs is closely associated with inhibiting the LSC-related anti-apoptotic axis,namely,reducing the expression of the LSC-related anti-apoptotic factor-HLF gene and increasing the expression of the downstream pro-apoptotic factor-NFIL3 gene.
引文
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[5] Park JW, Hong K, Kirpotin DB, et al. Anti-HER2 immunoliposomes: enhanced efficacy due to targeted delivery. Clin Cancer Res, 2002,8:1172-1181.
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[9] Lopes de Menezes DE, Pilarski LM, Allen TM. In Vitro and in Vivo Targeting of Immunoliposomal Doxorubicin to Human B-Cell Lymphoma. Cancer Res, 1998, 58:3320-3330.
[10]Sapra P, Allen TM. Improved outcome when B-cell lymphoma is treated with combinations of immunoliposomal anticancer drugs targeted to both the CD19 and CD20 epitopes. Clin Cancer Res,2004,10:2530-2537.
[11] Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med, 2002, 346:235-242.
[12] Matsumura Y, Gotoh M, Muro K, et al. Phase I and pharmacokinetic study of MCC-465, a doxorubicin (DXR) encapsulated in PEG immunoliposome, in patients with metastatic stomach cancer. Ann Oncol, 2004,15:517-25
[13] Sapra P, Moase EH, Ma J, et al. Improved therapeutic responses in a xenograft model of human B lymphoma (Namalwa) for liposomal vincristine versus liposomal doxorubicin targeted via anti-CD 19 IgG2a or Fab' fragments. Clin Cancer Res. 2004,10:1100-1111.
[14] Cheng WW, Das D, Suresh M, et al. Expression and purification of two anti-CD 19 single chain Fv fragments for targeting of liposomes to CD19-expressing cells. Biochim BiophysActa, 2007,1768: 21-29.
[15] Zhou Y, Drummond DC, Zou H, et al. Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells. J Mol Biol, 2007,371:934-947.
[16] Cheng WW, Allen TM. Targeted delivery of anti-CD 19 liposomal doxorubicin in B-cell lymphoma: a comparison of whole monoclonal antibody, Fab' fragments and single chain Fv. Control Release.2008,126:50-58.
[17] Lopes de Menezes DE, Pilarski LM, Belch AR, et al. Selective targeting of immunoliposomal doxorubicin against human multiple myeloma in vitro and ex vivo. Biochim Biophys Acta. 2000,1466:205-220.
[18] Harata M, Soda Y, Tani K, et al. CD19-targeting liposomes containing imatinib efficiently kill Philadelphia chromosome-positive acute lymphoblastic leukemia cells. Blood, 2004,104:1442-9.
[19] Ma J, Cao LR.Anti-CD19-liposomes encapsulated domain III of pseudomonas exotoxin in the targeting of human B lymphoma.Zhonghua Zhong Liu Za Zhi. 2003,25:36-38.
[20] Chiu GN, Edwards LA, Kapanen AI, et al. Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs. Mol Cancer Ther, 2007,6:844-855.
[21] Laginha K, Mumbengegwi D, Allen T. Liposomes targeted via two different antibodies: assay, B-cell binding and cytotoxicity. Biochim Biophys Acta. 2005, 1711:25-32.
[22] Lundberg BB, Griffiths G, Hansen HJ. Cellular association and cytotoxicity of doxorubicin-loaded immunoliposomes targeted via Fab' fragments of an anti-CD74 antibody. Drug Deliv,2007,14:171-175.
[23] de Kruif J, Storm G, van Bloois L, et al. Biosynthetically lipid-modified human scFv fragments from phage display libraries as targeting molecules for immunoliposomes. FEBS Lett. 1996,399:232-236.
[24] Orciani M, Cavaletti G, Fino V, et al. Exploiting CD38-mediated endocytosis for immunoliposome Intemalization. Anti-Cancer Drugs,2008,19:599-605.