發展對抗CHC之人源化抗體運用於胰臟癌之治療與造影

Development of anti-CHC humanized antibody for targeted drug delivery and imaging of pancreatic cancer

郭雅勛(Ya-Hsun Kuo)

指導教授 ： 吳漢忠

國立臺灣大學/牙醫專業學院/口腔生物科學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10900

摘要

胰臟癌的死亡率高居全球所有癌症的第五名，由於早期診斷上的困難，也缺乏有效的治療方式，導致胰臟癌是個致死率高、極度惡性的癌症，因此針對胰臟癌發展新的治療與診斷策略實屬當務之急。過去的研究顯示，網格重鏈蛋白（CHC）在胰臟癌細胞中呈現高度表現，並且扮演了促進腫瘤生長和腫瘤血管新生的角色。在本篇研究當中，我們更進一步發現，在內源性CHC表現量較低的癌細胞中，以轉染方式送進質體使CHC過度表現，會增進癌細胞移動以及轉移的能力。另一方面，研究也顯示在缺氧狀態下，細胞膜上的CHC會和缺氧誘發因子-1α （HIF-1α）在細胞質有直接的交互作用。基於以上的發現，我們希望發展以CHC為標的之藥物傳輸系統。由於先前的研究中，我們以融合瘤技術得到了一株能專一性辨認CHC蛋白的鼠源單株抗體；在本篇研究，我們將這株鼠源單株抗體的重鏈變異區與輕鏈變異區以一段短胜肽連結成單鏈抗體（scFv），保留其互補決定區域（CDR），而將抗體架構區域修飾成人源化序列，完成的人源化單鏈抗體命名為hPa65-2 scFv。我們發現hPa65-2 scFv不僅對胰臟癌細胞有極佳的鍵結能力，也能高度被細胞進行內化作用。我們更進一步發現將hPa65-2 scFv與微脂體進行鍵結，可以促進微脂體專一性辨認癌細胞，以及進一步進行內化作用。於藥物傳輸系統的應用上，可以發現帶有抗癌藥物的微脂體在鍵結了hPa65-2 scFv之後，對於人類胰臟癌細胞異體移植的免疫缺陷型小鼠有較好的療效。另一方面，在造影醫學的應用上，我們將鍵結有hPa65-2 scFv的量子點與沒有鍵結的量子點分別以靜脈注射的方式打入小鼠體內，進行活體螢光影響觀察，發現將hPa65-2 scFv鍵結上量子點能增加量子點辨認腫瘤細胞的能力，也提高量子點累積於腫瘤與正常器官的比值。綜合以上結果，hPa65-2 scFv這株專一性辨認CHC蛋白的單鏈抗體，不僅能應用於藥物傳輸系統，增加化療藥物的專一性，提升其治療效果，還可以結合造影系統，以協助胰臟癌患者的早期診斷。

關鍵字

胰臟癌 ； CHC ； HIF-1α ； 單鏈抗體 ； 藥物傳輸系統 ； 微脂體

並列摘要

Pancreatic adenocarcinoma is a highly malignant cancer with poor prognosis due to lack of effective therapies and diagnosis. Therefore, development of new strategies to improve treatment and diagnosis is urgently desirable. We have previously demonstrated that clathrin heavy chain (CHC) protein promotes tumor growth and angiogenesis in pancreatic cancer. In this study, we further found that CHC protein overexpression significantly increased cell migration and invasion ability. In the hypoxic state, CHC would bind with HIF-1α protein in cytoplasm. In order to develop CHC-targeted drug delivery for cancer therapy, we engineered the humanized scFv (single-chain fragment variable) antibody against CHC, which was derived from anti-CHC monoclonal antibody Pa65-2 and was named hPa65-2 scFv. We have verified that hPa65-2 scFv not only retained the ability to bind to pancreatic cancer cells efficiently, but it also exhibited high level of endocytosis activity. Conjugation of hPa65-2 scFv with liposomal SRB was able to improve binding and internalization efficiency of liposomes to tumor cells. We also found that hPa65-2 scFv-conjugated liposomes can enhance delivery of doxorubicin into cancer cells and increase its cytotoxic effect. In a tumor xenograft model, hPa65-2 scFv-conjugated liposomes were found to improve therapeutic efficacy of a chemotherapeutic drug. In order to develop targeting imaging in tumor-bearing mice, hPa65-2 scFv were conjugated with quantum dots. In vivo fluorescence imaging showed that scFv-conjugated quantum dots increased tumor uptake and improved tumor-normal tissue ratio compared to non-targeting quantum dots. We conclude that anti-CHC scFv has significant potential in targeted drug delivery and imaging for pancreatic cancer.

並列關鍵字

pancreatic cancer ； clathrin heavy chain ； HIF-1α ； scFv ； drug delivery systems ； immunoliposome

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