卵巢癌相关分子标志物的初步鉴定及新型靶向抗体的制备
发布时间：2018-10-09 13:08
【摘要】：研究背景：卵巢癌是妇科常见恶性肿瘤之一，也是病死率最高的妇科肿瘤。由于发病隐匿且缺乏有效的早期诊断方法，以致70%的患者确诊时已属晚期，5年存活率仅为20%-30%。目前主要的治疗方法是手术和化疗，但约60％～70％的患者在治疗中发生了耐药和复发转移。因此，寻找早期诊断手段及有效治疗方法，对提高卵巢癌患者的生存率至关重要。 CAI25是目前应用最广泛的检测卵巢肿瘤的分子标志物，但CA125水平在生理排卵期、子宫内膜异位症、盆腔炎症及子宫腺肌瘤等妇科良性疾病中也会升高，出现假阳性结果，可见其用于卵巢癌诊断的敏感度及特异度不佳。随着肿瘤分子生物学的发展，与卵巢癌相关的新的分子标志物不断问世，也为肿瘤的分子靶向治疗提供了依据。2012年NCCN指南中，推荐用于复发卵巢癌的靶向治疗药物只有抗血管生成的贝伐珠单抗。此外，针对表皮生长因子受体家族如HER1、HER2、HER3以及胰岛素样生长因子1受体（IGF1R）等分子的靶向治疗的药物也研究较多。其中，HER2是乳腺癌治疗的经典靶点，上市的药物有曲妥珠单抗(Trastuzumab)和帕妥珠单抗(Pertuzumab)。有研究表明，HER2在卵巢癌组织中高度表达，并且HER2和卵巢癌的发生、发展及不良预后相关。 目的：鉴定与卵巢癌密切相关的分子标志物，合理选择潜在的治疗靶点，通过构建大容量噬菌体抗体库，从中筛选人源优化抗体并鉴定新抗体的体内外功能，初步探讨新抗体抑制卵巢癌的作用机制。 材料与方法： 1、收集患有妇科良性疾病的女性血清样本68例，检测其血清HE4和CA125的水平，进而评价HE4在诊断卵巢癌中的特异性。 2、采用生物芯片的技术手段对HER2、HER3、IGF1R等分子在卵巢癌组织中的特异性表达及其与肿瘤分期的关系进行初步分析； 3、利用Cre-LoxP重组系统构建大容量表位定向抗体库，用于新抗体的筛选并鉴定新抗体的体内外功能； 4、利用pCMV抗体真核表达质粒，构建全抗表达载体，对抗体的抗原结合、亲和力、抗原表位及体内抑瘤等效应进行初步鉴定； 5、利用卵巢癌细胞系SKOV3，在体外鉴定新抗体对其迁移、凋亡、ADCC及信号转导等的影响。 结果：1、患有妇科良性疾病的女性血清中，HE4水平的增加低于CA125，表明HE4在诊断卵巢癌的特异性优于CA125。2、HER2、HER3、IGF1R等分子在卵巢癌组织中呈高表达并与肿瘤的分期密切相关，均可作为卵巢癌潜在治疗的靶点。3、基于HER2-pertuzumab的复合物结构及作用模式，利用Cre-LoxP重组系统建立了大容量表位定向抗体库，并从库中筛选出了1株潜在而亲和力提高的优化抗体，，表位没有发生漂移，且与抗原的结合能力及体内抑瘤能力均有增强。4、经真核表达纯化获得了M5抗体，M5能够发挥体内抑瘤效应，其机制可能在于：抑制细胞迁移、诱导细胞凋亡、发挥ADCC效应、阻断HER2下游信号的活化。
[Abstract]:Background: ovarian cancer is one of the most common gynecological malignancies and has the highest mortality. Because of the hidden disease and the lack of effective early diagnosis, 70% of the patients were diagnosed at the late stage, and the 5-year survival rate was only 20 to 30. Surgery and chemotherapy are the main treatments at present, but about 60% of the patients develop drug resistance and recurrence and metastasis. Therefore, finding early diagnosis and effective treatment is very important to improve survival rate of ovarian cancer patients. At present, CAI25 is the most widely used molecular marker for detecting ovarian tumors, but the level of CA125 also increases in the physiological ovulatory period, endometriosis, pelvic inflammation and uterine adenomyoma and other benign gynecological diseases, with false positive results. It can be seen that the sensitivity and specificity of the diagnosis of ovarian cancer is not good. With the development of tumor molecular biology, new molecular markers related to ovarian cancer are emerging, which also provide the basis for the molecular targeted therapy of tumor. In the 2012 NCCN guidelines, Only anti-angiogenic bevacizumab is recommended for targeted treatment of recurrent ovarian cancer. In addition, many drugs targeting epidermal growth factor receptor family such as HER1,HER2,HER3 and insulin-like growth factor 1 receptor (IGF1R) have been studied. Among them, trotozumab (Trastuzumab) and padytozumab (Pertuzumab). Are the classic targets for breast cancer treatment. Studies have shown that HER2 is highly expressed in ovarian cancer, and HER2 is associated with the occurrence, development and poor prognosis of ovarian cancer. Objective: to identify the molecular markers closely related to ovarian cancer, to select potential therapeutic targets reasonably, to screen human antibodies and to identify the functions of new antibodies in vitro and in vivo by constructing a large phage antibody library. To explore the mechanism of inhibition of ovarian cancer by new antibody. Materials and methods: 1. 68 female patients with benign gynecologic diseases were collected and their serum HE4 and CA125 levels were measured to evaluate the specificity of HE4 in the diagnosis of ovarian cancer. 2. The specific expression of HER2,HER3,IGF1R and its relationship with tumor staging in ovarian cancer tissues were analyzed by biochip technique. 3. Large capacity epitope oriented antibody library was constructed by Cre-LoxP recombination system. It can be used to screen new antibody and identify its function in vivo and in vitro. (4) using eukaryotic expression plasmid of pCMV antibody, the whole anti expression vector was constructed, and the antigen binding and affinity of the antibody were obtained. The antigenic epitopes and anti-tumor effects in vivo were preliminarily identified. (5) the effects of new antibodies on migration, apoptosis and signal transduction of ovarian cancer cell line SKOV3, were identified in vitro. Results the increase of serum he4 level in women with benign gynecologic diseases was lower than that of CA125,. The specificity of HE4 in the diagnosis of ovarian cancer was higher than that of CA125.2,HER2,HER3,IGF1R and was closely related to the stage of ovarian cancer. Based on the complex structure and action pattern of HER2-pertuzumab, a large capacity epitope directed antibody library was established by using Cre-LoxP recombination system, and a potential and enhanced affinity antibody was screened from the library. The epitope did not drift, and the binding ability to antigen and tumor inhibition in vivo were enhanced. The anti-tumor effect of M5 antibody M5 was obtained by eukaryotic expression and purification. The mechanism may be as follows: inhibiting cell migration and inducing cell apoptosis. The activation of downstream signal of HER2 was blocked by ADCC effect.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.31
本文编号：2259442
[Abstract]:Background: ovarian cancer is one of the most common gynecological malignancies and has the highest mortality. Because of the hidden disease and the lack of effective early diagnosis, 70% of the patients were diagnosed at the late stage, and the 5-year survival rate was only 20 to 30. Surgery and chemotherapy are the main treatments at present, but about 60% of the patients develop drug resistance and recurrence and metastasis. Therefore, finding early diagnosis and effective treatment is very important to improve survival rate of ovarian cancer patients. At present, CAI25 is the most widely used molecular marker for detecting ovarian tumors, but the level of CA125 also increases in the physiological ovulatory period, endometriosis, pelvic inflammation and uterine adenomyoma and other benign gynecological diseases, with false positive results. It can be seen that the sensitivity and specificity of the diagnosis of ovarian cancer is not good. With the development of tumor molecular biology, new molecular markers related to ovarian cancer are emerging, which also provide the basis for the molecular targeted therapy of tumor. In the 2012 NCCN guidelines, Only anti-angiogenic bevacizumab is recommended for targeted treatment of recurrent ovarian cancer. In addition, many drugs targeting epidermal growth factor receptor family such as HER1,HER2,HER3 and insulin-like growth factor 1 receptor (IGF1R) have been studied. Among them, trotozumab (Trastuzumab) and padytozumab (Pertuzumab). Are the classic targets for breast cancer treatment. Studies have shown that HER2 is highly expressed in ovarian cancer, and HER2 is associated with the occurrence, development and poor prognosis of ovarian cancer. Objective: to identify the molecular markers closely related to ovarian cancer, to select potential therapeutic targets reasonably, to screen human antibodies and to identify the functions of new antibodies in vitro and in vivo by constructing a large phage antibody library. To explore the mechanism of inhibition of ovarian cancer by new antibody. Materials and methods: 1. 68 female patients with benign gynecologic diseases were collected and their serum HE4 and CA125 levels were measured to evaluate the specificity of HE4 in the diagnosis of ovarian cancer. 2. The specific expression of HER2,HER3,IGF1R and its relationship with tumor staging in ovarian cancer tissues were analyzed by biochip technique. 3. Large capacity epitope oriented antibody library was constructed by Cre-LoxP recombination system. It can be used to screen new antibody and identify its function in vivo and in vitro. (4) using eukaryotic expression plasmid of pCMV antibody, the whole anti expression vector was constructed, and the antigen binding and affinity of the antibody were obtained. The antigenic epitopes and anti-tumor effects in vivo were preliminarily identified. (5) the effects of new antibodies on migration, apoptosis and signal transduction of ovarian cancer cell line SKOV3, were identified in vitro. Results the increase of serum he4 level in women with benign gynecologic diseases was lower than that of CA125,. The specificity of HE4 in the diagnosis of ovarian cancer was higher than that of CA125.2,HER2,HER3,IGF1R and was closely related to the stage of ovarian cancer. Based on the complex structure and action pattern of HER2-pertuzumab, a large capacity epitope directed antibody library was established by using Cre-LoxP recombination system, and a potential and enhanced affinity antibody was screened from the library. The epitope did not drift, and the binding ability to antigen and tumor inhibition in vivo were enhanced. The anti-tumor effect of M5 antibody M5 was obtained by eukaryotic expression and purification. The mechanism may be as follows: inhibiting cell migration and inducing cell apoptosis. The activation of downstream signal of HER2 was blocked by ADCC effect.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R737.31
【共引文献】
相关期刊论文 前4条
1 李武菊;;卵巢癌早期诊断及治疗分析[J];临床和实验医学杂志;2011年12期
2 尚丽新;赵雯;王心;;卵巢癌的诊治与预防[J];人民军医;2013年01期
3 冯秀银;张萍;;异黏蛋白在卵巢上皮性癌中的表达及临床意义[J];实用妇产科杂志;2013年11期
4 王静;陶健;袁超;;卵巢癌患者联合检测血清TK1、CA125的临床价值[J];中华全科医学;2014年06期
本文编号：2259442
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