靶向肺癌标记物Mesothelin的嵌合抗原受体T细胞体内体外抗肿瘤活性研究

发布时间：2018-05-19 04:39

本文选题：嵌合抗原受体 + 间皮素　；参考：《第二军医大学》2016年博士论文

【摘要】：肺癌发病率高,每年因肺癌死亡的人数常居所有恶性肿瘤之首,但因其病情隐匿,超过80%患者就诊时已经是肺癌晚期,丧失了手术机会。同时,肺癌又是一种免疫原性较弱的恶性肿瘤,在癌细胞进化过程中人类白细胞抗原(HLA-1)的表达减少甚至缺失,因而不被树突状细胞有效识别而逃逸机体免疫系统攻击。因此,寻找有效的治疗手段成为亟待解决的难题。近年来,利用嵌合抗原受体(chimeric antigen receptors,CARs)通过基因工程改造的T淋巴细胞进行的过继性细胞免疫疗法在肿瘤治疗研究,尤其是血液系统肿瘤治疗研究中取得了突破性进展,但对实体瘤的研究由于对已知靶点的抗原知之尚少而相对滞后。与其它常规的肿瘤免疫治疗方法相比,CAR-T治疗的效应T细胞的靶向性、杀伤性和活力都相对较高,同时还能打破肿瘤病灶部位微环境的免疫抑制以及缓解宿主的免疫耐受。CAR修饰赋予T细胞含有特定抗原的受体,允许T细胞靶向攻击潜在的任何肿瘤。随着重组DNA技术的不断发展以及对信号传导通路研究的不断深入,CAR分子的信号域也从刚开始第一代的单一靶向信号发展为包含CD28、CD137(4-1BB)、CD134(OX40)和ICOS等共刺激分子的多信号区域的二代、三代。近年来,CAR-T技术在包括肺癌在内的多种癌症的临床应用中显示出一定的抗肿瘤效果,特别是在白血病、淋巴瘤、黑色素瘤等恶性肿瘤的控制方面显示出较好的治疗优势。但是,与其它肿瘤免疫治疗一样,CAR-T治疗也存在一些问题与不足,包括脱靶效应,以及基因工程手段将重组CAR分子导入淋巴细胞所致的插入突变等风险。因此,寻找肿瘤细胞更特异的表面抗原、研发安全有效的转导技术等成为完善CAR-T技术的关键所在。肿瘤相关抗原-间皮素(Mesothelin,MSLN),一种由71-kDa的前体成熟转变成的40-kDa糖基磷脂酰肌醇锚定糖蛋白,存在于细胞表面。MSLN在间皮瘤、非小细胞肺癌、食道癌和和转移性三阴性乳腺癌等多种肿瘤组织中高表达,在正常组织中不表达或间皮组织中低表达,而成为备受关注的肿瘤细胞特异靶向抗原。且有研究报道MSLN可促进肿瘤恶性表型形成,MSLN阳性的患者肿瘤预后更差。此外,在正常胸膜,腹膜,心包膜等间皮组织中MSLN的表达较少,因此以间皮素为靶点来构建CAR-T为免疫细胞治疗提供了一个安全的选择。将MSLN作为肿瘤特异性抗原进行靶向治疗不仅限于CAR-T治疗这一种肿瘤免疫疗法,还有靶向MSLN的免疫重组抗毒素SS1P、结合到抗MSLN的抗体单链可变区和人的IgG1及к恒定区的嵌合单克隆抗体MORAb-009、结合MSLN的一个有效的DNA烷化剂MDX-1382(Medarex)等多种疗法。MSLN已经被证实在约30%-70%的肺癌组织中高表达。因此,MSLN可以作为靶标应用于肺癌的肿瘤免疫治疗中。基于国内外肺癌免疫治疗的研究现状,由于MSLN作为肿瘤抗原有其独特的优越性,我们研究靶向MSLN的嵌合抗原受体T细胞体内外抗肿瘤活性。我们采用基因工程的手段将靶向MSLN的鼠源抗体的可变区联合共刺激分子4-1BB及CD28的三代CARs分子载入到慢病毒体系,用构建成功的慢病毒感染分选出的T细胞,同时以未感染的T细胞为对照,通过流式实验检测重组CAR-T细胞表面MSLN CAR的表达,通过体外检测CAR-T对过表达MSLN肿瘤细胞株Hela细胞的杀伤能力确定重组CAR-T对MSLN靶向的能力。此外,我们构建了CHO-K1-Mesothelin重组细胞株,使CHO细胞表面过表达人源化的MSLN,模拟重组CAR-T对肿瘤细胞表面的MSLN靶向研究,同样也发现重组CAR-T对CHO-K1-Mesothelin重组细胞株有显著的杀伤能力,进一步验证了重组CAR-T靶向MSLN的能力。最后,将肺癌组织在免疫缺陷NPG小鼠中构建PDX模型,经尾静脉注射重组CAR-T细胞和正常T细胞检测重组CAR-T细胞体内杀伤肿瘤的能力,通过测量肿瘤体积变化判定重组CAR-T细胞对肿瘤的杀伤情况,结果发现在回输之后的10天内,瘤组织的生长受到明显抑制,瘤体积显著小于对照组,说明重组CAR-T细胞在回输10天内抑制了肺癌的发展,可有效杀伤肺癌细胞。综上所述,通过本实验研究,我们明确了具有靶向肿瘤特异抗原MSLN的重组CAR-T细胞能在体外杀伤过表达MSLN的肿瘤细胞,同时在小鼠的PDX肺癌模型中证实重组CAR-T细胞能控制肺癌的发展,降低肿瘤体积的增长。这些前期的体外实验以及体内的动物模型实验将为CAR-T技术在临床上应用于过表达MSLN的肺癌提供理论基础。
[Abstract]:The incidence of lung cancer is high, and the number of people dying of lung cancer is the first one of all malignant tumors. But because of its concealment, more than 80% patients have advanced lung cancer and lost the chance of operation. At the same time, lung cancer is a kind of malignant tumor with weak immunogenicity, and the expression of human leukocyte antigen (HLA-1) is reduced during the process of cancer cell evolution. Therefore, the search for effective treatment has become an urgent problem. In recent years, the adoptive cell immunotherapy using chimeric antigen receptors (CARs) through genetically engineered T lymphocytes is swollen. The research on tumor treatment, especially in the blood system tumor treatment, has made a breakthrough, but the study of solid tumors is relatively lagging behind the known target antigens. Compared with other conventional tumor immunotherapy methods, the targeting, killing and vitality of the CAR-T treated T cells are relatively high, and at the same time It is possible to break the immunosuppression of the microenvironment of the tumor site and to alleviate the host's immune tolerance.CAR modification endow the T cell with specific antigen receptor, allowing T cells to target any potential tumor. With the continuous development of the recombinant DNA technology and the continuous development of the signal transduction pathway, the signal domain of the CAR molecule is also from the rigid The first generation of single target signals developed into two and three generations of multi signal regions including CD28, CD137 (4-1BB), CD134 (OX40) and ICOS. In recent years, CAR-T technology has shown a certain anti swelling effect in the clinical application of various cancers including lung cancer, especially in leukemia, lymphoma, melanoma and other evil. The control of sexual tumors shows good therapeutic advantage. However, as with other tumor immunotherapy, CAR-T therapy also has some problems and deficiencies, including the Miss effect, and the risk of inserting the CAR molecule into the lymphocyte by gene engineering, so as to find a more specific surface antigen of the tumor cells, The key to improving CAR-T technology is the development of safe and effective transduction technology. The tumor associated antigen mesothelin (Mesothelin, MSLN), a 40-kDa glycosyl phosphatidyl inositol anchoring glycoprotein transformed from the precursor of 71-kDa, exists on the cell surface.MSLN in mesothelioma, non small cell lung cancer, esophagus cancer and metastatic three negative High expression in a variety of tumor tissues such as breast cancer, which is not expressed in normal tissues or in mesothelial tissue, has become a highly focused tumor cell specific target antigen. It has been reported that MSLN can promote the formation of malignant tumor phenotype and the prognosis of MSLN positive patients is worse. In addition, in normal pleura, peritoneum, and cardiac capsule, and other mesothelial groups The expression of MSLN in the fabric is less, so using mesothelin as a target to construct CAR-T provides a safe choice for immune cell therapy. Targeting MSLN as a tumor specific antigen is not limited to CAR-T therapy, a tumor immunotherapy, and a targeted MSLN immunized group of antitoxin SS1P, combined with a single chain of anti MSLN antibody. The mutable region and human IgG1 and the chimeric monoclonal antibody MORAb-009 of the constant region, combined with a variety of therapeutic.MSLN, such as an effective DNA alkanating agent MDX-1382 (Medarex) of MSLN, have been shown to be highly expressed in the lung cancer tissues of approximately 30%-70%. Therefore, MSLN can be used as a target for cancer immunotherapy in lung cancer. Based on the immunity of lung cancer at home and abroad The research status of the treatment, because MSLN has its unique superiority as a tumor antigen, we study the anti-tumor activity of the chimeric antigen receptor T cells targeted to MSLN in vivo and in vivo. We use genetic engineering to carry the three generation CARs molecules of the mutable co stimulatory molecule 4-1BB and CD28 to the slow virus system. The T cells, selected from the successfully constructed lentivirus infection, were used to detect the expression of MSLN CAR on the surface of the recombinant CAR-T cells by flow test, and the ability of CAR-T to target the targeting of Hela cells in MSLN tumor cell line in vitro was detected by flow test. In addition, we constructed C. HO-K1-Mesothelin recombinant cell line, which made the CHO cell surface overexpressed human derived MSLN, simulated the MSLN targeting of the recombinant CAR-T on the surface of the tumor cells, and also found that the recombinant CAR-T had a significant killing ability to the CHO-K1-Mesothelin recombinant cell line, further validates the ability of the recombinant CAR-T to target MSLN. Finally, the lung cancer tissue is removed from the tissue. The PDX model was constructed in the epidemic defect NPG mice. The recombinant CAR-T cells and normal T cells were injected into the tail vein to detect the ability to kill the tumor in the recombinant CAR-T cells. The tumor volume was determined by the measurement of the tumor volume. The results showed that the growth of the tumor tissue was obviously suppressed within 10 days after the retransmission, and the tumor body was obviously inhibited. The product was significantly smaller than the control group, indicating that recombinant CAR-T cells could inhibit lung cancer development in 10 days and effectively kill lung cancer cells. In this experiment, we identified that recombinant CAR-T cells with target tumor specific antigen MSLN can kill MSLN tumor cells in vitro and PDX lung cancer models in mice. It is confirmed that recombinant CAR-T cells can control the development of lung cancer and reduce the growth of tumor volume. In vitro experiments in vitro and animal model experiments in vivo will provide a theoretical basis for the clinical application of CAR-T technology to lung cancer over expression of MSLN.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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