【作者】 黄亮；

【导师】 周剑峰；

【作者基本信息】 华中科技大学 ， 血液病学， 2011， 博士

【摘要】 [目的]急性髓系白血病作为一种高度异质性的疾病,诱导失败和耐药复发是其治疗失败的主要原因。而白血病干细胞是急性髓系白血病发生、耐药和复发的根源,针对白血病干细胞的靶向治疗更有可能根治白血病。IL-3受体介导的信号传递是促进急性髓系白血病细胞增殖的原因之一；IL-3受体α链/CD123更是急性髓系白血病干细胞的标志之一,而正常造血干细胞表面表达水平低下。因此,制备能靶向杀伤白血病干细胞的抗CD123的单克隆拮抗抗体,可能具有潜在的治疗价值。[方法]第一部分：克隆IL3RA胞外区与人IgGFc重组,构建两种真核表达载体,即pG4Fc-3.3-hIL3RA SP+ECD和pYDll-hIL3RAECD,瞬时转染HEK-293F细胞,选择表达效率最佳的载体表达、纯化并鉴定IL3RA ECD/Fc融合蛋白,作为免疫原。同时,我们定向克隆IL3RA编码区构建另外两种真核表达载体,即hIL3RA CDS-pEGFP-N1和pcDNA3.1(+)-IL3RA CDS,前者用于制备稳定表达CD123的细胞株以供后续检测和筛选抗体,后者将作为另一种免疫方式免疫小鼠；为克服质粒免疫动物后血清抗体效价低的缺点,我们还重组包装了能表达CD123的腺病毒Ad-IL3RA,供质粒免疫后的加强免疫使用。第二部分：利用前面构建的真核表达载体hIL3RA CDS-pEGFP-Nl脂质体转染CHO细胞,经过流式分选、G418筛选和流式、Elisa和Western blot鉴定CD123的表达水平,获得能稳定高表达CD123的细胞株,供制备抗体时筛选抗体用。同时,以IL3RAECD/Fc融合蛋白、表达IL3RA的急性红白血痫TF-1细胞株和前述构建的重组真核表达载体pcDNA3.1(+)-IL3RA CDS质粒三种方式进行免疫,Elisa筛选血清抗体效价高的Balb/c小鼠,取其脾细胞与小鼠骨髓瘤SP2/O细胞融合,经过Elisa和间接流式挑选阳性孔,再进行数轮有限稀释和筛选,挑选出能分泌抗CD123单克隆抗体的杂交瘤细胞株,供后续功能试验用。第三部分：建立IL-3增殖拮抗实验和急性白血病免疫缺陷NOD/SCID小鼠异种移植模型,分别检测这11株抗体竞争性拮抗IL-3/IL3RA交联及IL-3的促增殖效应,介导ADCC效应抑制白血病CD34+CD38细胞归巢和CD45+细胞植入,降低肿瘤负荷并延长生存期的靶向治疗效应。[结果]除利用TF-1细胞免疫未能获得抗体以外,我们利用制备的重组蛋白和质粒两种抗原免疫,各获得了3株和8株能分泌抗CD123抗体的杂交瘤细胞株,并经Elisa和间接流式证实杂交瘤株所分泌抗体与IL3RA ECD/Fc融合蛋白、TF-1和8G9细胞表面CD123的亲和力,且质粒免疫获得的1株抗体在后续的IL-3增殖拮抗实验中被证实能阻断细胞膜表面IL-3/IL3RA的交联以及IL-3的促增殖效应,表明是一株拮抗抗体。为进一步验证治疗性抗体抗肿瘤功能,还建立并验证了急性白血病免疫缺陷NOD/SCID小鼠模型。[结论]利用质粒免疫、腺病毒加强的方式制备抗体效率最高,更易获得有功能的治疗性抗体。共获得11株能分泌抗CD123单克隆抗体的杂交瘤细胞株,通过IL-3增殖拮抗实验,从中筛选出1株能竞争性拮抗IL-3/IL3RA的交联及IL-3的促增殖效应的拮抗抗体5A2,尚需利用建立的模型验证5A2是否介导ADCC效应,抑制白血病CD34+CD38-细胞归巢和CD45+细胞植入,降低肿瘤负荷并延长生存期。更多还原

【Abstract】 Objective:Acute myeloid leukemia (AML) is a heterogeneous disease, treatment failure and relapse is the major cause for poor prognosis. Leukemic stem cells (LSCs) is the origin of disease persistence, drug resistance and relapse, so the LSCs targeted therapy may present as a curable treatment for AML. The signal transduction drived from interleukin-3 receptor involved in the stimulation of proliferation of AML blasts, and interleukin-3 receptor a chain (IL3RA)/CD123 is a marker for LSCs in AML, at variance with normal hematopoietic stem cells (HSCs) that are IL3RA-. So, the LSCs targeted, anti-CD123 antagonist monoclonal antibody may present as a promising therapy for AML.Methods:PartⅠ:To product and purify IL3RA ECD/Fc fusion protein as an immunogen, we cloned the extracellular domain (ECD) of CD123 to human IgG Fc and constructed two eukaryotic expression vectors, pG4Fc-3.3-hIL3RA SP+ECD and pYDll-hIL3RA ECD, then HEK-293F cells were transfected with these plasmids transiently after optimizing of the transfection conditions, finally we purified and characterized the generated fusion protein. On the other hand, we cloned coding sequence (CDS) of CD123 to construct the other two vectors, hIL3RA CDS-pEGFP-N1 and pcDNA3.1(+)-IL3RA CDS. The former will be used to generate a CD123 overexpression cell line for subsequent antibody screening; the later will be used as immunogen. An adenovirus for immunization boost is needed for the immunization with plsmid.Part II:To generate a CD123 overexpression cell line for antibody screeing, we transfected CHO cells with hIL3RA CDS-pEGFP-Nl and subjected to FACS sorting and G418 screening. The expression of generated cell lines were evaluated by FACS, Elisa and Western blot. At the same time, we immunized Balb/c mice with different methods, including IL3RA ECD/Fc fusion protein, CD123-expressing TF-1 AML cell line and eukaryotic vectors pcDNA3.1(+)-IL3RA CDS. After screening by Elisa, spleen cells of immunized mice with high serum titer were fused with myeloma SP2/0 cells. The positive wells were picked out by Elisa and indirect FACS. After several round of limited dilution and screening, hybridoma cell lines capable of secreting anti-CD123 antibodies were generated for subsequent functional assays.Part III:To evaluate the ability of generated hybridoma cell lines to secret antibodies and block the biding of IL-3/IL3RA and the following proliferation effect competitively, we established IL-3 antagonist assay. We also established several acute leukemia NOD/SCID xenotransplantation models to test whether these antibodies could inhibit the homing of CD34+CD38- and the engraftment of CD45+ leukemic cells, which will reduce tumor burden and improve survival.Results:Although immunization with TF-1 cells generated no antibodies, we still obtained 3 and 8 hybridoma cell lines by immunization with fusion protein and plasmid, respectively. Determined by Elisa and indirect FACS, all of the antibodies secreted from these 11 cell lines had affinity to IL3RA ECD/Fc and CD123 expressed on TF-1 and 8G9 cells, especially, one of them was confirmed as an antagonist antibody to block the biding of IL-3/IL3RA and the following proliferation effect. We also established several acute leukemia NOD/SCID xenotransplantation models to test the anti-cancer property of these antibodies.Conclusion:Immunized with plasmid and boosted with adenovirus is a very efficient way to gain therapeutic antibody with certain function. In the 11 hybridoma cell lines capable of secreting anti-CD123 antibody, one named 5A2 was picked out for its ability to block the biding of IL-3/IL3RA and the subsequent proliferation effect competitively. The remaining work is to evaluate whether 5A2 could evoke ADCC to inhibit the homing of CD34+CD38-and the engraftment of CD45+ leukemic cells, which will decrease tumor burden and improve survival.更多还原