【作者】 李洋；

【导师】 华树成；

【作者基本信息】 吉林大学 ， 内科学， 2011， 博士

【摘要】 利用基因工程操作技术对抗CEA单链抗体T84.66进行了二硫键改构,设计并人工合成了抗CEA scdsFv基因序列,并通过柔性连接肽将SEA基因序列融合在抗CEAscdsFv末端,此基因序列命名为SC-C/SEA。将上述核苷酸序列克隆入原核表达载体pET28a(+),构建了原核表达质粒pET-SC-C/SEA。将原核表达质粒转化感受态大肠杆菌BL21,利用异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达,并对诱导时间、IPTG浓度等表达条件进行优化,从而获得目的蛋白SC-C/SEA。利用SDS-PAGE和Western blotting对SC-C/SEA的表达进行了鉴定。利用亲合层析方法对SC-C/SEA进行复性和初步纯化。利用免疫荧光结合流式细胞术分析、MTT等方法探讨了SC-C/SEA对A549细胞的识别和结合能力及其特异性；分析了SC-C/SEA对体外培养的A549细胞增殖的影响；检测了重组蛋白SC-C/SEA对PBMC中CTL功能的作用。实验结果表明,SC-C/SEA可特异性识别A549细胞(CEA阳性)并与之结合,对HeLa细胞(CEA阴性)无此活性。SC-C/SEA对A549细胞的结合活性存在一定的剂量和时间依赖关系,其最佳作用条件为20μl作用4h; SC-C/SEA基本对体外培养的细胞无明显毒性作用；另外,PBMCCTL活性的检测结果表明,SC-C/SEA能够刺激针对A549细胞的特异性CTL反应,且其作用具有一定的效靶比依赖性。本研究利用所构建和表达的重组蛋白SC-C/SEA、SC-C和SEA对肺癌实体肿瘤模型进行探索性治疗研究。结果表明,SC-C/SEA能有效延长肺癌实体模型动物的平均生存期、提高生存几率。另外,SC-C/SEA具有减缓肿瘤组织生长速度的作用。对免疫指标的检测表明,SC-C/SEA能够非特异性的促进某些细胞因子的分泌,且具有刺激增强抗肿瘤CTL和NK活性的作用。更多还原

【Abstract】 The carcinoembryonic antigen (CEA), known as an important clinical tumor marker, is a highly glycosylated oncofetal glycoprotein containing 50% carbohydrates with a molecular weight of about 180-200 kDa. CEA is a tumor-associated antigen uptrgulated in the majority of colon cancers, non-small cell lung cancers and half of all breast cancers. On the other hand, CEA is also present in some normal tissues. A lot of research demonstrated that, in normal tissues, CEA was mainly localized on the luminal surface of the single layer of columnar epithelial cells lining the upper parts of the crypts and was not directly in contact with blood flow or tissue fluid. While, in tumor tissues, CEA was localized on all sides of the cell membranes and was directly facing blood flow or tissue fluid. All these features made it an attractive target molecule for antibody-directed diagnosis, gene therapy and immunotherapy.Although monoclonal antibody was considered one of the preferred forms of cancer immunotherapy, many other methods have been proved more effective. Liu Y et al produced a dual functional protein that exhibited both tumor specific binding and killing activities by fusing the tumor-specific apoptosis-inducing molecule Apoptin to an anti-CEA single chain antibody. Alternatively, one study was to localize interleukin-2 (IL-2) to cancer cells by joining the IL-2 to the antibodies specific for tumor antigens. These studies indicated that antibody-directed targeting of the antitumoral molecule could alleviate the toxic side effects while enhancing the proteins deposition at tumor sites. Genetically engineered fusion proteins basing on tumor specific antibody have received more emphasis.The single-chain disulfide-stabilized Fv antibody (scdsFv), comprised of the variable heavy domain (VH) and the variable light domain (VL), is the smallest antibody fragment containing a complete antigen binding site. The engineered stabilizing disulfide bond in scdsFv can overcome the problem of aggregatjon at high concentrations of the basic single-chain Fv antibody (scFv). In comparison with the much larger IgG, scdsFv fragments have lower retention times in non-target tissues, a more rapid blood clearance and better tumor penetration in vivo. The use of labeled scdsFv that target the tumor-associated antigen has been described extensively.The selection of human anti-CEA single-chain antibody fragment (scFv) is a key step toward the development of new antitumoral agent designed for immunotherapy based on CEA. The engineered scFv of anti-CEA T84.66 antibody used for this research has been studied extensively since its discovery. Phase I clinical trials have demonstrated that the engineered scFv of anti-CEA T84.66 antibody was rapid tumor uptake, high tumor activity, fast blood clearance and suitable for clinical practice.Staphylococcal enterotoxin A (SEA), known as superantigens (SAgs) because of the ability to recognize Vβregion of T-lymphocyte receptors, is a one of the bacerial superantigen, involving in autoimmune and toxic shock disorders, that activates immune responses and induces production of various cytokines. Previous studies have indicated that SEA was the most potent stimulator of T cells and tumor necrosis factor (TNF). Furthermore, SEA stimulates T cells mainly in histocompatiblity complex classⅡrestricted manner and induces high levels of cytokines, such as TNF, IL-2 and interferon-y. These characteristic of SEA might be useful in therapy for regulating immune responses.In present study, we construted a chimeric protein, designated as SC-C/SEA that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A (SEA) to C-terminus of an anti-CEA single-chain disulfide-stabilized Fv antibody (scdsFv). The SC-C/SEA proteins were expressed in Escherichia coli (E. coli), refolded and purified on an immobilized Ni2+ affinity chromatography column. SDS-PAGE and Western blotting revealed that the target protein was well-expressed. We demonstrated by immunofluorescence assays that SC-C/SEA could bind specifically to human lung carcinoma cells (A549), but almost not to human uterine cervix (HeLa). We also observed the anti-tumor activity of the recombinant proteins in vivo. The solid tumor model and the in situ model were established. The mean survival, the tumor growth, the suppression rate, the NK activity, the CTL activity and the cytokine levels of the animal models were evaluated. The results showed that the recombinant proteins inhibited the lung cell growth effectively. Although the injection of the recombinant adenoviruses did not lead to complete elimination of the tumors, effective inhibition was observed in the established solid tumor model and the in situ model. In summary, SDS-PAGE and Western blotting analysis demonstrated that the recombinant protein was well expressed. Immunofluorescent and CTL assays indicated that SC-C/SEA could bind to CEA-positive cells specifically and induce CTL predominance. The SC-C/SEA protein produced in the work can be developed for potential use in CEA-targeted cancer immunotherapy.更多还原