- 學位論文
開發針對淋巴瘤的雙功能抗體(anti-CD3 x anti-CD20)改善免疫治療效果
To develop the bispecific antibody (anti-CD3 x anti-CD20) against lymphoma for improving immunotherapy
摘要
惡性淋巴瘤是一種發生在淋巴組織的實體瘤，其中10%是Hodgkin's lymphoma (HL)，其餘都是non- Hodgkin's lymphoma (NHL)。在先前的研究中發現CD20會大量表現在惡性B細胞上，因此研究家們開發出針對CD20的單株抗體，例如Ofatumumab (OFA)，目前免疫治療在對NHL治療可達到很好的療效，例如bispecific T-cell engager (BiTE)和CART，但是在治療上還是面臨一些副作用，CART療法由於體內CART過度活化造成嚴重的副作用，BiTE因為是二段式結合的方式，無法準確定量在體內T細胞與BiTE的結合多寡，所以BiTE必須給予高劑量來維持藥物效果，因此我們的策略是建構一可同時辨識T細胞(anti-CD3)及腫瘤細胞(anti-CD20)之雙功能抗體，治療方式維將患者T細胞萃取出後，加入雙功能抗體與T細胞在體外進行結合並活化(One-step mixing)，透過這樣的方式監控T細胞的使用量並降低藥物劑量。我們以基因工程技術建構出雙功能抗體質體(αCD3 X αDNS、αCD3 X αCD20)，transient transfection至293T細胞確認質體DNA可以在哺乳動物細胞表現蛋白，並利用西方墨點法急流是細胞一確認雙邊功能具備功能性，在透過Ni Sepharose High Performance純化抗體，並利用SDS-PAGE確認純化後的抗體是高純度的，利用PHA-M和IL-2刺激活化人類周邊血單核球細胞(peripheral blood mononuclear cell，PBMC)評估抗體的功能性。結果顯示質體DNA可以在293T細胞生產並且蛋白質具有功能性，此策略若成功可以有效用於改善惡性淋巴瘤的病人上。
並列摘要
Lymphomas is a kind of solid tumor of the immune system. The frequency of Hodgkin's lymphoma (HL) is around 10% in all lymphomas, and the remaining is non-Hodgkin's lymphoma (NHL). The immunotherapy in NHL is an evolving modality. The previous studies have found that CD20 molecule is overexpressed in B-NHL, so the monoclonal antibodies aguinst CD20 are designed for immunotherapy, like Ofatumumab (OFA). Now in clinical, the NHL would benefit from the further application of immunotherapy. The mechanism of the CAR T cells and the bispecific T-cell engager (BiTE) have all demonstrated efficacy in NHL, but these therapies still have some defect and toxicity that cause patients discomfort and death. The CART cell in the patient body is overexpression and the BiTE is two-step mix for the t cell and tumor cell, so we can not quantitative BiTE and t cell, so we want to improve the therapy defect for enhancing the therapeutic efficacy. In our topic, we will develop the bispecific antibody that stable antibody and binding on the T cells that calls Armed-T in vitro, and it can reduce the dose-escalation and quantity the t cell. We prepared the BsAb constructions and transiently transfected the gene to 293T cells to confirm the expressions of the BsAbs. We also evaluated the binding efficiency of BsAb using flow cytometry. Using the Ni Sepharose High Performance (Ni column) to purify the BsAb and re-checking the BsAb binding which armed on the T cell . In this study, we confirmed the expression and function of the BsAbs in 293T cell successfully. The next, we used the Ni column to purify protein, and the SDS-PAGE showed the BsAb was purified by ni column and had the high quality. In the results of the flow cytometry show T cells had been stimulated with PHA-M and IL-2 in day 6 that had high activity, and the BsAbs can target on the T cells. We confirmed the BsAb can binding on PBMC. In the future, maybe can used on malignant lymphoma patients.