卵巢癌是婦科癌症死亡原因的首位，由於它缺乏早期的症狀及診斷的方法，因此在婦女癌症中有極高的致死率。其中第二大類型的卵巢亮細胞癌 (OCCC) 其預後通常比其他類型的卵巢癌更差，原因在於OCCC 對於化學治療的抗藥性。由於缺乏良好的診斷手段，病患多半發現於晚期，因此發展一個有用的生物標記及治療方針，對於卵巢亮 細胞癌的患者而言是十分重要的。實驗室先前的研究上發現到FXYD2 (Na/ K ATPase的輔助蛋白) 在卵巢亮細胞癌相比於其他種類的卵巢癌和正常人具有異常的高度表現，同時與病人的預後有強烈的 相關性。並且在異植腫瘤的老鼠模式上 (Xenograft animal model) 藉由Cardiac glycoside (心臟病用藥) 治療降低Na/ K ATPase 活性可以有效的抑制卵巢亮細胞癌的生長，提升老鼠的存活。
近年來，免疫療法治療癌症的發展非常活躍，其中嵌合抗原受體T細胞療法chimeric antigen receptor T- cells (CAR-T) 治療手段具有強大的潛力。CAR-T治療是藉由體外基因工程的方式將能夠辨認腫瘤上特定胜肽的單株抗體嵌合到T細胞上，使T細胞具有更大的機會追蹤到腫瘤細胞，且活化的T細胞能夠產生更多的T細胞增加毒殺腫瘤細胞的機會。而CAR-T治療的想法在B細胞急性淋巴性白血病上證實了其治療潛力。利用B細胞淋巴瘤上專一性表現CD19的特性，將Anti-CD19的抗體序列藉由基因轉殖 (transduction) 的方式嵌合到T細胞產生Anti-CD19 CAR-T成功的治療了B細胞急性淋巴性白血病的病患。而後許多研究團隊希望將CAR-T治療的想法運用到固體腫瘤上 (solid tumor)。然而固體腫瘤相比於白血症這樣的血液型癌症具有更多的困難需要克服，包含了腫瘤細胞的微環境 (tumor microenvironment) 會抑制免疫細胞毒殺腫瘤細胞的活性以及T細胞浸潤到腫瘤細胞的難度。其中最嚴重的問題在於CAR-T治療的潛在毒性，細胞激素風暴 (cytokine release syndrome, CRS)。CRS最主要產生的原因在於當CAR-T細胞辨認到腫瘤細胞並且活化後會不斷地釋放許多細胞激素，例如TNF-α，這些細胞激素會促使免疫細胞釋放IL-6，而IL-6會持續的活化免疫細胞，最終導致了IL-6的暴增，這樣的CRS在Anti-CD19 CAR-T治療B細胞急性淋巴性白血病上也有一樣的情況發生，且在臨床治療上發生機率高達8成。CRS的產生會導致高燒、休克，嚴重者甚至會導致死亡。
本實驗中希望藉由靶向FXYD2，設計出專一性抗FXYD2的anti-FXYD2 CAR-T細胞，來治療OCCC的病患，並以原位腫瘤動物模式 (Orthotopic model) 去測試OCCC之治療效果。此外，以往轉導到T細胞的CAR分子皆以DNA為主，以達到治療的持久性，然而也導致了CRS的不可逆性。因此本研究在往後實驗上希望將CAR分子改為mRNA的層級，使得CAR-T細胞具有時效性，藉由控制輸入到病人體內的CAR-T細胞量來抑制CRS的情況。

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy. Due to the lack of early diagnosis methods, it has been a very high mortality rate in female cancer. According to the gene expression and histomorphology, it consists of 5 different histologic including serous (62%), clear cell (17%), endometrioid (15%), mucinous (4%) and other (2%). The second-largest subtype of ovarian cancer, clear cell carcinoma (OCCC), usually has a worse prognosis than other subtypes due to OCCC resistance to chemotherapy. Because of the lack of effective diagnosed strategies, most women were found and diagnosed at an advanced stage. Therefore, developing a useful biomarker and treatment is urgent for OCCC-bearing patients. Previous studies have shown that the FXYD2 (Na+/K+ ATPase accessory protein) is highly expressed in OCCC and has a strong correlation with the prognosis of patients. In the Xenograft animal model, the reduction of Na+/K+ ATPase activity after Cardiac glycoside treatment can effectively inhibit the growth of tumor cells and improve the survival rate of mice.
Immunotherapy has received considerable attention in cancer treatment, while chimeric antigen receptor T-cells (CAR-T) therapy has proven the potential of cancer therapy. CAR-T therapy is to chimeric monoclonal antibodies that can recognize specific antigen on tumor cells act as a receptor to T cells through in vitro genetic engineering. CAR-T cells can track tumor cells to achieve the goal of killing tumor cells. Based on the specific feature of CD19 in B-cell lymphoma, Anti-CD19 CAR-T cells have shown the response in the patient bearing acute lymphocytic leukemia. Despite the success in treating hematological malignancies, it still have lots of challenges on the solid tumor through CAR-T therapy. For example, in the solid tumor treatment, the tumor microenvironment (TME) can inhibit the activity of immune cytotoxicity cause to T cells infiltration into tumors is difficulty. The most challenge is how to reduce the side effect of CAR-T therapy, cytokine release syndrome (CRS). The most challenge is how to reduce the side effect of CAR-T therapy, cytokine release syndrome (CRS). The main reason for CRS is the continuously release many cytokines, such as TNF-α when CAR-T cells recognize tumor cells. These cytokines were stimulated immune cells and release IL-6 more than1000 folds as compare with the general situation. In addition, CRS not only existence in solid tumor therapy but also in the situation of B-cell acute lymphoblastic leukemia with Anti-CD19 CAR-T treatment. The occurrence in clinical treatment is more than 80%, and then lead to high fever, shock, and even death in severe cases.
In this study, we aim to develop a CAR-T cell therapy for OCCC treatment. FXYD2 as the targeting molecule to design a specific anti-FXYD2 CAR-T cell to evaluate the therapeutic effect in OCCC cell lines. Besides, according to the previous study, the CAR molecules transduced to T-cells were mainly DNA level to achieve treatment durability, but it also led to the irreversibility of CRS. Therefore, we will transfer the CAR molecular to mRNA level to make CAR-T cells have the half-life characteristic. Based on the conclusions, we can decrease the CRS and on-target off-tumor toxicity via mRNA CAR-T cells. We believe that the FXYD2 has the potential to become the therapeutic target of CAR-T cells, provide a new strategy for OCCC patients.

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