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 P. pastoris 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.

Keywords: Fc glycosylation; Pichia; endoglycosidase; glycoengineered antibodies; trastuzumab.

Fig. 1.

Construction and expression of recombinant trastuzumab in P. pastoris and characterization of glycans on purified glycoproteins using liquid chromatography–mass spectrometry analysis. (A) Construction of expression plasmid. (B) KanMX replacement cassette for homologous recombination of yeast chromosomal integration module. (C) Characterization of recombinant trastuzumabs produced by WT and YKO P. pastoris strains by using SDS/PAGE. (D) Glycan profiling of recombinant trastuzumabs produced by WT and YKO yeast strains. 5′HS/3′HS, 5′/3′-end homologous sequence; 6xHis, the polyhistidine tag; Δ, gene deletion; heavy chain, the heavy chain OR of trastuzumab; KanMX, kanamycin (G418) resistance gene; Light chain, the light chain OR of trastuzumab; mAOX1 promoter, modified alcohol oxidase 1 promoter region; rHC, recombinant heavy chain; rHC(t), truncated form of recombinant heavy chain; rLC, recombinant light chain; TT, the alcohol oxidase 1 transcriptional terminator region; Zeo(R), zeocin resistance gene.

Fig. 2.

ENGase catalyzed hydrolysis of complex and high-mannose type glycoproteins. (A) Characterization of glycan hydrolysis activities on Herceptin. (B) Characterization of glycan hydrolysis activities on RNaseB. (Upper) Western blotting. (Lower) Glycan MS.

Fig. 3.

Glycosylation remodeling of recombinant trastuzumab to prepare homogeneous glycoforms with well-defined glycan of sialyl-2,6-linkage for optimal effector functions. GlcNAc-trastuzumab, recombinant trastuzumab with single GlcNAc in the Fc region; M₅-trastuzumab, recombinant trastuzumab with Man5 glycans in the Fc region as a major glycoform; SCT, sialylated biantennary complex type glycan; SGP, sialylated biantennary complex type glycopeptide; SHM-trastuzumab, recombinant trastuzumab with super high mannose glycans in the Fc region.

Fig. 4.

Characterization of selected ENGases with regard to the deglycosylation activity on recombinant rHer/ΔOCH1. Two micrograms purified recombinant rHer/ΔOCH1 was digested with 100 ng ENGase at 37 °C for 4 h and profiled by SDS/PAGE.

Fig. 5.

EndoE-catalyzed deglycosylation of high-mannose glycans on recombinant rHer/ΔOCH1. (A) Characterization of EndoE-treated rHer/ΔOCH1. Two micrograms purified rHer/ΔOCH1 was digested with 100 ng EndoE at 37 °C for 4 h and profiled by SDS/PAGE. (B) Characterization of EndoE-treated rHer/ΔOCH1 by using liquid chromatography–mass spectrometry. The ratio showed in the y axis was relative glycan amount on the Fc region of recombinant rHer/ΔOCH1.

Fig. 6.

EndoS2 (WT) catalyzed transglycosylation of trastuzumab. Transglycosylation efficiency was evaluated by SDS/PAGE analysis (Upper). The ratio of protein bands on SDS/PAGE was calculated by ImageQuant TL software and presented as the amount of IgG (%) in the y axis of the lower plot. G, GlcNAc; HC, heavy chain; Her, trastuzumab; LC, light chain; SCT, α2,6-sialylated biantennary complex type glycan.