卵巢癌相關分子標志物的初步鑒定及新型靶向抗體的制備
發(fā)布時間：2018-10-09 13:08
【摘要】：研究背景：卵巢癌是婦科常見惡性腫瘤之一，也是病死率最高的婦科腫瘤。由于發(fā)病隱匿且缺乏有效的早期診斷方法，以致70%的患者確診時已屬晚期，5年存活率僅為20%-30%。目前主要的治療方法是手術和化療，但約60％～70％的患者在治療中發(fā)生了耐藥和復發(fā)轉(zhuǎn)移。因此，尋找早期診斷手段及有效治療方法，對提高卵巢癌患者的生存率至關重要。 CAI25是目前應用最廣泛的檢測卵巢腫瘤的分子標志物，但CA125水平在生理排卵期、子宮內(nèi)膜異位癥、盆腔炎癥及子宮腺肌瘤等婦科良性疾病中也會升高，出現(xiàn)假陽性結果，可見其用于卵巢癌診斷的敏感度及特異度不佳。隨著腫瘤分子生物學的發(fā)展，與卵巢癌相關的新的分子標志物不斷問世，也為腫瘤的分子靶向治療提供了依據(jù)。2012年NCCN指南中，推薦用于復發(fā)卵巢癌的靶向治療藥物只有抗血管生成的貝伐珠單抗。此外，針對表皮生長因子受體家族如HER1、HER2、HER3以及胰島素樣生長因子1受體（IGF1R）等分子的靶向治療的藥物也研究較多。其中，HER2是乳腺癌治療的經(jīng)典靶點，上市的藥物有曲妥珠單抗(Trastuzumab)和帕妥珠單抗(Pertuzumab)。有研究表明，HER2在卵巢癌組織中高度表達，并且HER2和卵巢癌的發(fā)生、發(fā)展及不良預后相關。 目的：鑒定與卵巢癌密切相關的分子標志物，合理選擇潛在的治療靶點，通過構建大容量噬菌體抗體庫，從中篩選人源優(yōu)化抗體并鑒定新抗體的體內(nèi)外功能，初步探討新抗體抑制卵巢癌的作用機制。 材料與方法： 1、收集患有婦科良性疾病的女性血清樣本68例，檢測其血清HE4和CA125的水平，進而評價HE4在診斷卵巢癌中的特異性。 2、采用生物芯片的技術手段對HER2、HER3、IGF1R等分子在卵巢癌組織中的特異性表達及其與腫瘤分期的關系進行初步分析； 3、利用Cre-LoxP重組系統(tǒng)構建大容量表位定向抗體庫，用于新抗體的篩選并鑒定新抗體的體內(nèi)外功能； 4、利用pCMV抗體真核表達質(zhì)粒，構建全抗表達載體，對抗體的抗原結合、親和力、抗原表位及體內(nèi)抑瘤等效應進行初步鑒定； 5、利用卵巢癌細胞系SKOV3，在體外鑒定新抗體對其遷移、凋亡、ADCC及信號轉(zhuǎn)導等的影響。 結果：1、患有婦科良性疾病的女性血清中，HE4水平的增加低于CA125，表明HE4在診斷卵巢癌的特異性優(yōu)于CA125。2、HER2、HER3、IGF1R等分子在卵巢癌組織中呈高表達并與腫瘤的分期密切相關，均可作為卵巢癌潛在治療的靶點。3、基于HER2-pertuzumab的復合物結構及作用模式，利用Cre-LoxP重組系統(tǒng)建立了大容量表位定向抗體庫，并從庫中篩選出了1株潛在而親和力提高的優(yōu)化抗體，，表位沒有發(fā)生漂移，且與抗原的結合能力及體內(nèi)抑瘤能力均有增強。4、經(jīng)真核表達純化獲得了M5抗體，M5能夠發(fā)揮體內(nèi)抑瘤效應，其機制可能在于：抑制細胞遷移、誘導細胞凋亡、發(fā)揮ADCC效應、阻斷HER2下游信號的活化。
[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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.31
本文編號：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.
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R737.31
【共引文獻】
相關期刊論文 前4條
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本文編號：2259442
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