靶向卵巢癌TEM1的特异性成像和免疫治疗
发布时间:2018-04-22 11:31
本文选题:卵巢癌 + 肿瘤内皮细胞标记物1 ; 参考:《吉林大学》2014年博士论文
【摘要】:肿瘤内皮细胞标记物1(TEM1)作为肿瘤血管标记物被认为能够观察肿瘤血管的再生。我们的前期工作显示:与正常卵巢组织比较,卵巢癌组织的肿瘤血管内皮细胞过表达TEM1,因此,推测TEM1可能是一个肿瘤血管治疗的标记物。同时,我们使用酵母抗体库筛选分离出人的TEM1特异性单链抗体(scFv78),并发现TEM1能结合到人和鼠的TEM1的胞外区(aa.324-390),其亲和指数Kd值约2nM,表明其亲和力较差。另外,scFv78分子量小,容易经肾脏排泄,血浆半衰期短,限制了其在早期特异性诊断和治疗等方面的应用。 本实验为了提高TEM1特异性抗体的亲和力与延长其在血液中的半衰期。我们构建了一系列新的scFv78多价抗体衍生物,包括二聚体Fc融合蛋白(来自hu IgG1)(78Fc),四聚体CH2融合蛋白(78CH2),二聚体CH3融合蛋白(78mb)和二聚体的CH1-铰链区融合蛋白(78F(ab')2),以便从中发现温度稳定和血清稳定性更好以及亲和力更高的抗体;通过瞬时转染293F细胞并且对培养上清进行亲和纯化获得这些蛋白;利用活细胞ELISA对这些蛋白进行亲和力的分析;同时,在动物体内对上述5个蛋白的血液代谢动力学进行检测;并利用近红外荧光成像技术对筛选出的最佳候选蛋白进行了体内成像检测,利用细胞杀伤实验和成管实验,观察其体外选择性地杀伤TEM1阳性表达细胞的效果。实验结果显示:与我们前期工作发现的scFv78相比,本实验构建的78Fc亲和力(Kd=0.14±0.01nM)增加了约15倍;动物体内代谢动力学结果表明78Fc的慢相半衰期约5.1小时;体内功能分析表明78Fc不针对Naive鼠的正常器官,而只是针对体内TEM1+肿瘤;近红外荧光成像技术结果显示78Fc在鼠体内只靶向TEM1阳性表达的细胞;最后78Fc-MMAE可以在体外选择性地杀伤TEM1阳性表达细胞。 综上所述,我们可以初步认为,通过将scFv78和Fc段融合后,提高了该蛋白的稳定性及亲和力,血液动力学结果表明78Fc有相对长的血清半衰期,可以用来做成像的探针。体内近红外荧光成像技术,体外细胞杀伤实验与成管实验与也证实其具有较高的特异性。因此,我们认为TEM1是一个成像与治疗的候选基因,可能为卵巢癌和其它癌症的靶向诊断与治疗提供新的思路;而78Fc可以为卵巢癌的早期诊断和治疗提供可能。
[Abstract]:Tumor endothelial cell marker (Tem 1) is considered to be able to observe tumor vascular regeneration. Our previous work showed that tumor vascular endothelial cells in ovarian cancer tissues overexpressed TEM1 compared with normal ovarian tissues. Therefore, we speculated that TEM1 might be a marker of tumor vascular therapy. At the same time, we used yeast antibody library to screen and isolate human TEM1 specific scFv78, and found that TEM1 could bind to the extracellular region of TEM1 in human and mouse, and its affinity index (KD) was about 2 nm, which indicated that the affinity was poor. In addition, scFv78 has small molecular weight, easy excretion through kidney and short plasma half-life, which limits its application in early specific diagnosis and treatment. The purpose of this study was to improve the affinity of TEM1 specific antibodies and prolong their half-life in blood. We have constructed a series of new scFv78 polyvalent antibody derivatives. It includes dimer FC fusion protein (from Hu IgG1, CH2 fusion protein, tetramer CH2 fusion protein, dimer CH3 fusion protein, and dimer CH1-hinge fusion protein) and dimer CH1-hinge region fusion protein, so as to find antibodies with better temperature stability, better serum stability and higher affinity between the two fusion proteins, including the fusion protein Fc from Hu IgG1, the fusion protein from tetramer CH2, the fusion protein from dimer CH3, and the fusion protein from the CH1-hinge region of dimer. These proteins were obtained by transient transfection of 293F cells and affinity purification of the culture supernatant. The affinity of these proteins was analyzed by living cell ELISA. The best candidate protein was detected by near infrared fluorescence imaging in vivo. The effect of selective killing of TEM1 positive cells in vitro was observed by cell killing test and tube forming experiment. The results showed that the affinity of 78Fc constructed in this experiment was increased by about 15 times compared with the scFv78 found in our previous work, and the metabolic kinetics of 78Fc in vivo showed that the slow phase half-life of 78Fc was about 5.1 hours. In vivo functional analysis showed that 78Fc was not directed at normal organs of Naive mice, but only on TEM1 tumors in vivo, near infrared fluorescence imaging showed that 78Fc only targeted TEM1 positive cells in mice. Finally, 78Fc-MMAE can selectively kill TEM1 positive cells in vitro. In conclusion, we can preliminarily conclude that by fusion of scFv78 and FC, the stability and affinity of the protein are improved, and the hemodynamic results show that 78Fc has a relatively long serum half-life and can be used as an image probe. In vivo near infrared fluorescence imaging, in vitro and in vitro cell killing experiments and tube-forming experiments and also confirmed that it has a high specificity. Therefore, we believe that TEM1 is a candidate gene for imaging and treatment, which may provide a new idea for the targeted diagnosis and treatment of ovarian cancer and other cancers, while 78Fc can provide the possibility for early diagnosis and treatment of ovarian cancer.
【学位授予单位】:吉林大学
【学位级别】:博士
【学位授予年份】:2014
【分类号】:R737.31
【共引文献】
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