利用酵素法修飾酵母菌表現之重組蛋白上之醣分子以製備均相化抗體

Abstract

近年來開發以單株抗體作為治療癌症，抗發炎及防禦病原體等之標靶藥物深受矚目; 然而，抗體Fc部位上的醣體組成對其與各種免疫細胞間的作用力具有相當大之影響。目前市面上以哺乳動物細胞作為宿主表達出來之抗體，除了其Fc部位上帶有非均相化的醣分子，且生產過程易受到病毒污染，純化費時，價格亦相對昂貴，因此，開發具大量生產均相化、且結構明確的抗體表達系統為亟待解決之課題。本研究利用醣化基因OCH1缺陷之甲醇誘導型畢赤酵母菌表現trastuzumab抗體，所產出之抗體Fc上的醣分子以五顆甘露糖鏈結 (Man5) 為主; 接著，我們從CAZymes資料庫中篩選並以大腸桿菌E. coli表達出十種新的內切糖苷酶 (Endoglycosidase)，作為後續抗體醣化修飾之酵素，且這些新發現的酵素可替代現有之內切糖苷酶EndoA與EndoH，及轉醣酶EndoS。此外，EndoE與EndoP兩種酵素不僅可利用雙觸鏈結寡糖鏈 (bi-antennary oligosaccharide chains)結構的醣分子作為抗體上Fc醣修飾的醣體來源，亦有催化三觸及四觸鏈結寡糖鏈 (tri- and tetra-antennary oligosaccharide chains) 結構的醣分子之潛力。本研究提供醣修飾及優化醣蛋白的系統性方法，並能提升IgG抗體之功效。

Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Since the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody expression in mammalian hosts exhibits heterogeneous glycosylation patterns and is relatively expensive, new efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAb with desirable glycoforms. The methylotrophic yeast Pichia pastoris that produces glycoproteins with high-mannose N-glycans has recently been engineered to express therapeutic glycoproteins. In this study, we demonstrate that glycosylation remodeling of the monoclonal antibody trastuzumab expressed in glycoengineered P. pastoris, through de-glycosylation by endoglycosidases identified from the CAZymes database, and transglycosylation using glycans with stable leaving group will generate a homogeneous antibody designed to optimize the effector functions. The ten newly identified recombinant bacterial endoglycosidases are complementary to existing endoglycosidases (EndoA, EndoH, EndoS); and two of which can even accept sialylated tri- and tetra-antennary glycans as substrates. This study provides a new platform for use to modulate and optimize the functions of glycoproteins, including the effector functions of IgG antibodies.