Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation

Abstract

Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Because 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 production is relatively expensive, efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAbs with desirable glycoforms. In this study, we demonstrate that the mAb trastuzumab expressed in glycoengineered can be remodeled through deglycosylation by endoglycosidases identified from the Carbohydrate Active Enzymes database and through transglycosylation using glycans with a stable leaving group to generate a homogeneous antibody designed to optimize the effector functions. The 10 newly identified recombinant bacterial endoglycosidases are complementary to existing endoglycosidases (EndoA, EndoH, EndoS), two of which can even accept sialylated tri- and tetraantennary glycans as substrates.

Mining Data From Plasma Cell Differentiation Identified Novel Genes for Engineering of a Yeast Antibody Factory

“…Furthermore, it will be essential to generate antibodies with proper N-glycan structures. Unless using post-production in vitro glycosylation (Wei et al, 2008;Liu et al, 2018), it needs to be ensured that glycoengineering strategies are compatible with production optimized strains.…”

“…et al, 2009;Nasab et al, 2013;Laukens et al, 2015;Piirainen et al, 2016). Besides approaches modifying the N-glycosylation pathway in vivo, also enzymatic in vitro transglycosylation of yeast produced Fc-fragments and full-length antibody was demonstrated as a feasible option (Wei et al, 2008;Liu et al, 2018). However, major other cellular obstacles still need to be overcome, in particular the limited capacity of S. cerevisiae to produce and secrete recombinant proteins and foremost the structurally complex full-length antibodies.…”

Glycosylation as an Important Regulator of Antibody Function

“…Myšia myelómová bunková línia YB2/ 0 dokáže produkovať rekombinantné protilátky s nízkym ob sahom corefukozylácie, čo môže mat za následok až 50násobne vyššiu ADCC aktivitu oproti produkcii v CHO bun kách [38]. Takisto kvasinky nedokážu vy tvárať fukozylované štruktúry, ale zato tvoria hlavne vysokomanózové štruk túry, ktorých profil sa líši v závislosti na kmeni [86,87]. Vďaka genetickej modi fikácii dokážu byť pre produkciu tera peutických Ig využité aj rastliny, kde boli vypnuté enzýmy tvoriace imunogénne glykoformy [88].…”

Milestones in Bioorganic Chemistry

“…These efforts not only stimulated his interest in carbohydrate chemistry, but also resulted in research on practical systems for cofactor regeneration or studies on enzyme engineering. He also worked on many challenging areas of medical relevance like on the development of antibacterial and antiviral medications, for example, by exploring the therapeutic potential of aminoglycosides and RNA as a potential drug target, on the synthesis of glycoengineered antibodies or on investigating the immunostimmulatory potential of glycolipids . He still is a very active researcher, so there is certainly more to come.…”