本文選題：嵌合抗原受體 + 間皮素　； 參考：《第二軍醫(yī)大學》2016年博士論文

【摘要】：肺癌發(fā)病率高,每年因肺癌死亡的人數常居所有惡性腫瘤之首,但因其病情隱匿,超過80%患者就診時已經是肺癌晚期,喪失了手術機會。同時,肺癌又是一種免疫原性較弱的惡性腫瘤,在癌細胞進化過程中人類白細胞抗原(HLA-1)的表達減少甚至缺失,因而不被樹突狀細胞有效識別而逃逸機體免疫系統(tǒng)攻擊。因此,尋找有效的治療手段成為亟待解決的難題。近年來,利用嵌合抗原受體(chimeric antigen receptors,CARs)通過基因工程改造的T淋巴細胞進行的過繼性細胞免疫療法在腫瘤治療研究,尤其是血液系統(tǒng)腫瘤治療研究中取得了突破性進展,但對實體瘤的研究由于對已知靶點的抗原知之尚少而相對滯后。與其它常規(guī)的腫瘤免疫治療方法相比,CAR-T治療的效應T細胞的靶向性、殺傷性和活力都相對較高,同時還能打破腫瘤病灶部位微環(huán)境的免疫抑制以及緩解宿主的免疫耐受。CAR修飾賦予T細胞含有特定抗原的受體,允許T細胞靶向攻擊潛在的任何腫瘤。隨著重組DNA技術的不斷發(fā)展以及對信號傳導通路研究的不斷深入,CAR分子的信號域也從剛開始第一代的單一靶向信號發(fā)展為包含CD28、CD137(4-1BB)、CD134(OX40)和ICOS等共刺激分子的多信號區(qū)域的二代、三代。近年來,CAR-T技術在包括肺癌在內的多種癌癥的臨床應用中顯示出一定的抗腫瘤效果,特別是在白血病、淋巴瘤、黑色素瘤等惡性腫瘤的控制方面顯示出較好的治療優(yōu)勢。但是,與其它腫瘤免疫治療一樣,CAR-T治療也存在一些問題與不足,包括脫靶效應,以及基因工程手段將重組CAR分子導入淋巴細胞所致的插入突變等風險。因此,尋找腫瘤細胞更特異的表面抗原、研發(fā)安全有效的轉導技術等成為完善CAR-T技術的關鍵所在。腫瘤相關抗原-間皮素(Mesothelin,MSLN),一種由71-kDa的前體成熟轉變成的40-kDa糖基磷脂酰肌醇錨定糖蛋白,存在于細胞表面。MSLN在間皮瘤、非小細胞肺癌、食道癌和和轉移性三陰性乳腺癌等多種腫瘤組織中高表達,在正常組織中不表達或間皮組織中低表達,而成為備受關注的腫瘤細胞特異靶向抗原。且有研究報道MSLN可促進腫瘤惡性表型形成,MSLN陽性的患者腫瘤預后更差。此外,在正常胸膜,腹膜,心包膜等間皮組織中MSLN的表達較少,因此以間皮素為靶點來構建CAR-T為免疫細胞治療提供了一個安全的選擇。將MSLN作為腫瘤特異性抗原進行靶向治療不僅限于CAR-T治療這一種腫瘤免疫療法,還有靶向MSLN的免疫重組抗毒素SS1P、結合到抗MSLN的抗體單鏈可變區(qū)和人的IgG1及к恒定區(qū)的嵌合單克隆抗體MORAb-009、結合MSLN的一個有效的DNA烷化劑MDX-1382(Medarex)等多種療法。MSLN已經被證實在約30%-70%的肺癌組織中高表達。因此,MSLN可以作為靶標應用于肺癌的腫瘤免疫治療中�；趪鴥韧夥伟┟庖咧委煹难芯楷F狀,由于MSLN作為腫瘤抗原有其獨特的優(yōu)越性,我們研究靶向MSLN的嵌合抗原受體T細胞體內外抗腫瘤活性。我們采用基因工程的手段將靶向MSLN的鼠源抗體的可變區(qū)聯合共刺激分子4-1BB及CD28的三代CARs分子載入到慢病毒體系,用構建成功的慢病毒感染分選出的T細胞,同時以未感染的T細胞為對照,通過流式實驗檢測重組CAR-T細胞表面MSLN CAR的表達,通過體外檢測CAR-T對過表達MSLN腫瘤細胞株Hela細胞的殺傷能力確定重組CAR-T對MSLN靶向的能力。此外,我們構建了CHO-K1-Mesothelin重組細胞株,使CHO細胞表面過表達人源化的MSLN,模擬重組CAR-T對腫瘤細胞表面的MSLN靶向研究,同樣也發(fā)現重組CAR-T對CHO-K1-Mesothelin重組細胞株有顯著的殺傷能力,進一步驗證了重組CAR-T靶向MSLN的能力。最后,將肺癌組織在免疫缺陷NPG小鼠中構建PDX模型,經尾靜脈注射重組CAR-T細胞和正常T細胞檢測重組CAR-T細胞體內殺傷腫瘤的能力,通過測量腫瘤體積變化判定重組CAR-T細胞對腫瘤的殺傷情況,結果發(fā)現在回輸之后的10天內,瘤組織的生長受到明顯抑制,瘤體積顯著小于對照組,說明重組CAR-T細胞在回輸10天內抑制了肺癌的發(fā)展,可有效殺傷肺癌細胞。綜上所述,通過本實驗研究,我們明確了具有靶向腫瘤特異抗原MSLN的重組CAR-T細胞能在體外殺傷過表達MSLN的腫瘤細胞,同時在小鼠的PDX肺癌模型中證實重組CAR-T細胞能控制肺癌的發(fā)展,降低腫瘤體積的增長。這些前期的體外實驗以及體內的動物模型實驗將為CAR-T技術在臨床上應用于過表達MSLN的肺癌提供理論基礎。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R734.2

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