基于急性髓系白血病细胞CD123抗体介导的主动靶向免疫脂质体的构建

There are still problems on treatment of acute myeloid leukemia(AML) due to low cure rate,severe side effects and relapse. CD123 is an antigen specifically expressed on AML cells and its stem cells,which may serve as a potential site for therapeutic intervention to improve even eradicate AML.Firstly, we plan to construct a PEGylated immunoliposome(ILP) modified by CD123 antibody targeting to the AML cells.Secondly,we will quantify the CD123 on both preotein and gene expression level using multicolor flow cytometry and polymerase chain reaction technique to find out the subtype with the highest CD123 expression in order to confirm the most appropriate target cells,named as subtype Mx.Thirdly,a post-insertion method will be applied to prepare the ILP.Targeting efficiency and competitive binding assay as evaluation index,various targeting antibody fragment,antibody expression densities and PEG expression densities will be optimized to obtain the best formula targeting to the Mx.Finally,daunorubincin loaded PEGylated ILP will be prepared and then served to test in vitro cytotoxicity with MTT method.Soft agar colony formation assay and secondary transplant study will be carried out to evaluate in vivo antitumor efficacy on the AML grafted NOD/SCID mice model.It should be noted that the PEGylated ILP mediated by CD123 antibody targeting to the AML cells(stem cells)will be constructed for the first time in this project.It is supposed to develop new ideas and pave a new way for tretment of AML/AMLSC.

急性髓系白血病（AML）目前仍存在临床治愈率低、毒副反应大及易复发等难题。CD123是存在于AML及其干细胞（LSC）表面的特异性抗原，为改善甚至根治AML提供了潜在的治疗干预位点。本课题拟以CD123为靶点，构建抗CD123抗体修饰的PEG化免疫脂质体(ILP）。采用多色流式细胞分选术与聚酶链式反应从蛋白和基因水平分析AML各亚型CD123抗原的表达量，以此确定适宜的AML靶细胞亚型；采用后插入法构建ILP,以靶向效率与竞争结合试验结果为评价指标，考察靶向抗体类型、抗体及PEG链密度等因素，优选最佳的ILP配方；以柔红霉素为模型药物，制备载药ILP，以MTT法考察其对靶细胞的抗癌药效，软琼脂集落形成及二次移植试验考察其在AML移植的NOD/SCID 模型小鼠体内的抗癌效果。项目将首次构建基于抗AML细胞表面CD123抗体介导的主动靶向给药系统，为治疗AML/AMLSC拓展新思路与途径。

急性髓系白血病（AML）目前仍存在临床治愈率低、毒副反应大及易复发等难题。CD123是存在于AML及其干细胞（LSC）表面的特异性抗原，是有效的AML治疗靶点。本课题以CD123为靶点，构建抗CD123抗体修饰的PEG化免疫脂质体(ILP）。采用流式细胞仪分析AML各亚型CD123抗原的表达量，确定适宜的AML靶细胞亚型为THP-1（M5型）与KG-1a（AML干祖细胞系）；采用薄膜分散－超声法及硫酸铵梯度法制备PEG化载药脂质体。在此基础上，成功制备了抗CD123 mAb免疫脂质体，得到L-CD123-ILP（0.06%）和H-CD123-ILP（0.14%）两种抗体修饰密度不同的脂质体。对不同细胞摄取脂质体的差异以及CD123修饰的免疫脂质体对AML细胞的杀伤作用进行了比较研究。结果表明，对于CD123表达较高的THP-1与KG-1a细胞，其对CD123单抗修饰的脂质体的摄取显著高于未修饰组，且摄取量随抗体修饰密度增加而增大。反之，对于CD123表达较低的HL-60细胞，CD123修饰与否对细胞摄取量则无显著影响。预先加入游离CD123单抗，可显著抑制肿瘤细胞对免疫脂质体的摄取，提示CD123单抗及其修饰的脂质体系通过抗体介导的内吞作用实现对肿瘤细胞的靶向递药。肿瘤细胞增殖抑制与细胞摄取研究也呈现一致的结果，与未经修饰组相比，CD123单抗修饰的载DNR的脂质体对THP-1、KG-1a细胞具有更强的增殖抑制作用，该结果应与受体介导的内吞作用使进入肿瘤细胞内的药物量增加有关。同时，与低密度抗体修饰组相比，高密度抗体修饰脂质体组也显著增强了其对肿瘤细胞的杀伤作用。 药代动力学研究结果表明，与DNR游离药物相比，CD123单抗修饰的载药脂质体显著延长了原药在大鼠体内的生物半衰期，有望获得更优的体内抗肿瘤药效。. 项目首次构建了基于AML细胞表面CD123抗体介导的PEG化免疫脂质体，初步阐释了CD123抗体修饰靶向AML细胞的影响因素及其作用机制，为科学、合理构建抗体介导的主动靶向AML细胞给药系统提供了实验依据，为治疗AML/AMLSC拓展了新思路与给药途径。