【作者】 唐碧霞；

【导师】 何维； 崔莲仙；

【作者基本信息】 中国协和医科大学 ， 免疫学， 2006， 博士

【摘要】 人类MHCⅠ类链相关基因A（MHC classⅠchain-related A,MICA）是Tom Spies等人于1994年发现的、位于人第六号染色体上MHC-Ⅰ类区域的非经典HLA-Ⅰ类基因家族成员,具有高度多态性。MICA蛋白是一种细胞应激分子,研究表明,它在正常组织中表达量很少,仅在肠道上皮组织表达量稍高,但是在多种肿瘤细胞,尤其是上皮源性的肿瘤细胞表面表达量明显上调,可以通过与表达在NK细胞,γδT细胞,CD8⁺αβT细胞表面的NKG2D受体相结合而被效应细胞识别,介导上述细胞对肿瘤细胞的杀伤作用。在本实验室的前期工作中,我们已经从卵巢癌细胞系SKOV3中提取总RNA,通过RT-PCR克隆了MICA的全长cDNA。利用pET原核表达系统分别表达了含有MICA胞外结构域α1α2α3的融合蛋白MICAα1-3和胞外结构域α1-α2的融合蛋白MICAα1-2,并制备了抗MICAα1-2的单克隆抗体786、10C6、1287,证明它们均可以与细胞、组织表面的MICA蛋白特异性结合。在上述工作基础上,本研究工作进一步将MICA作为肿瘤相关抗原（tumor associated antigen,TAA）靶向性蛋白,利用其相应的鼠源单克隆抗体作为体内分子探针,研究MICA在临床肿瘤早期诊断和治疗中的应用前景。本论文工作主要研究内容包括三部分:（一） ^99mTc标记抗MICA特异性抗体10C6在卵巢癌定位诊断的研究利用ELISA技术测定抗rMICAα1-2单克隆抗体786、10C6、1287的亲和力,从中挑选出亲和力最高的抗体10C6。用直接标记法将^99mTc标记到抗体10C6分子上,利用细胞结合实验检测标记后抗体的活性,采用^99mTc示踪技术观察标记抗体在荷人卵巢癌细胞SKOV3裸鼠的体内分布及肿瘤摄取情况,并用γ相机观察全身显像情况,并实时检测标记抗体在小鼠血液和全身的清除速率。此外,还用NBS法将抗体10C6与¹³¹I连接,观察标记抗体在荷人卵巢癌细胞SKOV3裸鼠体内肿瘤的摄取情况,并将两种放射性核素标记的抗体在体内的显像结果作一比较。研究结果提示,MICA可以视为一种新的TAA,作为肿瘤靶向抗原,在肿瘤的早期诊断及治疗中有一定的应用前景。由于^99mTc的γ射线能量不高并且射线成份较纯,所以和¹³¹I相比,更适合用于肿瘤短期内的显像诊断。（二）高亲和力抗MICA特异性抗体的筛选为筛选到高亲和力抗MICA单克隆抗体,我们重新利用原核表达的重组蛋白rMICAα1-2制备了一系列的特异性抗MICA单克隆抗体。通过ELISA、流式细胞检测技术（flow cytomery,FCM）对抗体进行了筛选及特异性的鉴定。然后通过间接ELISA法,测得6株单抗的相对亲和力,再进一步利用非竞争性ELISA方法测定单克隆抗体7B12的亲和力。高亲和力抗MICAα1-2抗体7B12的获得为进一步的体内诊断定位研究提供了有力的工具。（三） ^99mTc标记抗MICA特异性抗体片段7812 Fab’在卵巢癌定位诊断的研究将纯化的抗体7B12用胃蛋白酶酶切纯化后,采用直接标记法将^99mTc标记到抗体片段F（ab’）2上,得到^99mTc-Fab’。采用^99mTc示踪技术观察标记抗体片段在荷人卵巢癌细胞SKOV₃裸鼠的体内分布及肿瘤摄取情况,并用γ相机观察全身显像情况,并实时检测标记抗体在小鼠血液和全身的清除速率。研究结果表明,和以往文献报道相一致,小分子的^99mTc-Fab’在体内的血液清除速率明显加快,在肾脏的潴留量大大超过其它脏器。但是本实验中^99mTc-Fab’肿瘤的靶向定位效果并不显著,这可能是由于由完整抗体的二价分子变成Fab’一价分子后,抗体效价降低有关,也可能是由于核素偶联制备过程中造成抗体片段亲和力的下降以及7B12抗体本身的特性所造成的。本研究的创新点在于:（1）通过放射性核素标记抗体在荷瘤小鼠体内分布代谢实验及显像实验,证实肿瘤相关抗原MICA蛋白,在肿瘤的早期诊断和治疗中可以作为靶向蛋白;（2）通过体内外实验,证实抗MICA单克隆抗体10C6和7812有一定的临床应用前景。更多还原

【Abstract】 MICA is a member of non-classical HLA-I gene family located in the MHC-I region on No. 6 chromosome, which was found by Tom Spies et al. in 1994. The gene is polymorphic. MICA protein is a cellular stress molecule, which is expressed little on the surface of normal tissue, while some amount on the cellular surface of the gastrointestinal tract. Its expression was also documented on the surface of many kinds of tumor cells, especially the epithelial tumor cells, such as in many carcinomas of the lung, breast, kidney, ovary, prostate and colon. By interacting with the NKG2D receptor on the surface of NK cell,γδT cell and CD8⁺ T cell, MICA can be recognized by those effect cells, resulting activation of the killing function of those cells.In the previous work, we obtained recombinant MICAα1-2, and the corresponding monoclonal antibody （mAb） 7B6, 10C6 and 12B7. Those mAbs could combine with native MICA on the cells and tissues specifically. Based on those results, we studied whether MICA could be recarded as a kind of TAA, targeted by its MAbs in vivo. Radionuclides ^99mTc and ¹³¹I were selected as labels. Our work provided the possibility of MICA as a target for tumor imaging and therapy on the early stage of disease. Furthermore, the corresponding anti-MICA mAbs 10C6 and 7B12 had the potential application in clinic. This paper included three parts:1. The study of ^99mTc-mAb 10C6 for the diagnosis in nude mice bearing MICA-positive tumorThe affinities of 7B6, 10C6 and 12B7 were determined by ELISA. 10C6, which was with the highest affinity, was labeled with ^99mTc by the direct labeling method. The biodistribution and the uptake of tumor tissue were detected. Gamma camera was used for the whole body image. At the same time, the clearance of the radiolabeled mAb in the whole body and the blood was detected at the appointed time. Furthermore, mAb 10C6 was labeled with ¹³¹I by NBS, in order to compare the imaging effect of different radionuclides. The results showed that MICA could be regarded as a TAA, which might be used as a target for tumor imaging and therapy at early stage. And for its lower energy and relatively pureγradiation, ^99mTc was considered as a more suitable imaging reagent for tumor in short term.2. The selection of anti-MICA mAb with high affinityrMICAα1-2 was expressed and purified. Then a series specific anti-MICA mAbs were obtained as follows: After immunized for 4 times with purified rMICAal-2, the mice were sacrificed and spleen cells were isolated in sterile condition. The spleen cells were hybridized with mouse myeloma SP2/0 cells through routine cell hybridization method. Positive clones were screened by conditional cultivation with HAT. Finally, hybridized myeloma cell clone 7B12 excreting monoclonal antibody with high affinity was selected by indirect ELISA, Western blot and flow cytometry. And its K value was obatianed by noncompetitive ELISA method.3. The study of ^99mTc-mAb 7B12 Fab’ fragment for the diagnosis in nude mice bearing MICA-positive tumorThe F（ab’）₂ of IgG 7B12, which was obtained by enzyme-digestion with pepsin, was labeled with ^99mTc directly. The biodistribution and the uptake of tumor tissue were detected. Gamma camera was used for the whole body image. At the same time, the clearance of the radiolabeled mAb fragment in the whole body and the blood was detected at the appointed time. The results showed that ^99mTc-Fab’ had lower tumor targeting than the whole IgG, but had a better blood clearance, which might be related with its lower avidity, biochemical character and the labeling procedure with ^99mTc.In this paper, innovative ideas included the following points: primarily, as a TAA, MICA was firstly proposed as a target for tumor imaging and therapy at early stage of diseases. Anti-MICA mAbs 10C6 and 7B12 have the potential application in clinic. And we provided a procedure of selecting mAb with high affinity in a relative shortterm.更多还原