摘要:將具有療效作用的大分子藥物以標靶性輸送至腫瘤組織是現今藥物遞送的挑戰之一。已進入臨床試驗的蛋白質藥物--基因合成精胺酸去亞胺酸脢 (recombinant arginine deiminase ,rADI) 用於治療肝癌與黑色素腫瘤,然而文獻指出,有部分肝癌細胞株 (如HepG2) 顯示對基因合成精胺酸去亞胺酸脢 (rADI) 的抗藥性。本實驗室先前的研究成果顯示,利用無毒性的穿膜載體HBHAc可有效增加基因合成精胺酸去亞胺酸脢 (rADI)遞送進入具抗藥性的癌細胞,因而增加基因合成精胺酸去亞胺酸脢 (rADI) 的毒殺作用。HBHAc是25 個胺基酸所組成的短片段胜肽,取自結核菌(Mycobacterium tuberculosis) 表面抗原蛋白中的肝素結合凝血附著素(heparin-binding haemagglutinin adhesion,HBHA) 的一小片段。由本實驗室先前的研究指出此段HBHAc具有穿膜作用的性質,且本實驗室已將此片段進行攜帶大分子,例如蛋白質或核酸,用於細胞遞送能力的研究。本計畫擬將依上述成果以及已建立之技術,設計雙重修飾的基因合成精胺酸去亞胺酸脢 (rADI),使之具有細胞內化及腫瘤標的作用。此載體設計為以HBHAc/半乳糖 (galactose) 增加細胞內化作用,運用主動 (galactose) 及被動標靶(enhanced permeability and retention, EPR) 的設計,已將蛋白藥品有效送至肝癌細胞內。實驗中所需的蛋白質都將由本實驗室已建立的蛋白質重組及純化技術製造。第一年的計畫目標是為了解此雙重修飾系統的作用效率、機制及安全性,將選用綠螢光蛋白 (EGFP) 作為模式蛋白,以於細胞實驗中,藉由螢光表現了解遞送過程中蛋白質的完整性。了解相關的機制作用後,第二年的計畫中,將以具療效的基因合成精胺酸去亞胺酸脢 (rADI)為模式蛋白,基於第一年的研究成果在蛋白質上修飾galactose及HBHAc,另鍵結樹枝狀聚合物dendrimer以利被動標的至腫瘤組織,並對修飾後的精胺酸去亞胺酸脢 (rADI)於體外進行作用效率、機制及安全性研究。第三年的實驗計畫,將利用前兩年研究最佳化的雙重修飾系統,於動物體內進行藥物活性、抗腫瘤作用、生物分佈和安全性的相關研究。藉由此三階段的研究計畫有助於了解此雙修飾系統應用於綠螢光蛋白 (EGFP) 及精胺酸去亞胺酸脢 (rADI) 於體外及體內的遞送效果及機制。期望藉由此創新設計的雙重修飾系統增加精胺酸去亞胺酸脢 (rADI) 的遞送,使之專一性標的並有效性內化進入腫瘤細胞,以減低非專一性所造成的副作用。此雙重修飾系統可望提供蛋白質藥物遞送的新選擇,使之同時具有腫瘤標的作用及增加腫瘤組織內化的作用。
Abstract: It is a challenge for the delivery of a therapeutic macromolecular in order to optimize the activity of anti-tumor and tumor targeting. Recombinant arginine deiminase (rADI), an arginine-depriving protein, has been used for the treatment of hepatoma and melanoma in clinical trials. However, the resistance of rADI in some hepatoma cell lines, such as HepG2, was reported. From our preliminary results, effectively delivery of rADI intracellularly can restore the rADI-sensitivity in rADI-resistant cancer cells by a nontoxic carrier (HBHAc), a fragment from the surface-exposed protein heparin-binding haemagglutinin adhesion (HBHA) from Mycobacterium tuberculosis. HBHAc, a 25 mer peptide, is studied in our laboratory for its ability as a carrier for macromolecules, such as proteins and nucleic acids based molecules. Dual modifications of rADI are proposed in this study in order to have the functions, cellular internalization and tumor targeting. Therefore, functional targeting via galactose receptor and passive targeting by enhanced permeability and retention (EPR) effect are used for the modification of interested protein in our study design. All the interested proteins are produced and purified by recombinant technology in our laboratory. In the first year, we would like to understand the effect, mechanism, and safety of a dual modified protein. The experiments would be performed in cultured cell experiments by using enhanced green fluorescence protein (EGFP) as a delivered protein. EGFP itself can be easily be detected and only the intact EGFP can be detected by its emitting fluorescence. After we understand the effective design of the protein modifications, the strategy would be applied into the second year plan for the therapeutic protein, rADI. The rADI would be modified by galctose/HBHAc according to the results from the first year. The dendrimer-based rADI is designed to ensure the EPR effect as well. The effect, mechanism, and safety of the modified rADI would be further evaluated in vitro. After we optimize the modifications of rADI according to the results from the second year, we would like to test the efficacy and safety of the modified protein in vivo. Different experiments are designed for the observation of biological activity, anti-tumor efficacy, bio-distribution, and safety as well in mice. At the end of the 3-year study, the characteristics and functionalities of the designed carrier should be investigated by delivering an enhanced green fluorescence protein (EGFP) and rADI, respectively. After this comprehensive study, rADI may be delivered specifically into cancer tissues by the newly designed deliver system, and subsequently increase the efficacy in anti-tumor activity and decrease the systemic adverse effects. A useful strategy applied for protein delivery would be rendered in order to increase the cell uptake and tumor targeting.