Fig 1.
(A) Protocol of chemoenzymatic synthesis using ENG’ase and glycosynthase. (B) Diagram of the homogeneous glycosylated anti-Her2 mAb with M3 (mAb-M3), G0 (mAb-G0), G2 (mAb-G2), and A2 (mAb-A2).
Fig 2.
MALDI QIT-TOF MS spectrum of Bz-labeled glycopeptides from anti-Her2 mAbs produced in silkworm cocoon.
Fig 3.
ENG’ase activity of the anti-Her2 mAbs (a; endoS, b; endoD, c; endoH, d; endoM, e; endoLL).
(Blue bar represents glycopeptides without ENG’ase hydrolysis; red bar represents the remaining glycopeptides with ENG’ase hydrolysis; y-axis indicates each individual glycoform ratio to total glycoform content; % represents total cleaved glycopeptide ratio by ENG’ase hydrolysis.)
Fig 4.
a) SDS-PAGE analysis of lane 1; anti-Her2 mAb from silkworm cocoon before IEX column chromatography (52.5 and 53.7 kDa as heavy chains (H), 29.8 kDa as light chains (L)), lane 2; anti-Her2 mAb from silkworm cocoon after IEX column chromatography (mAb; 53.7 kDa (H), 29.8 kDa (L)), the homogeneous glycosylated anti-Her2 mAb with M3 after IEX column (mAb-M3, lane 3; 53.5 kDa (H), 29.8 kDa (L)), G0 after IEX column chromatography (mAb-G0, lane 4; 53.7 kDa (H), 29.8 kDa (L)), G2 after IEX column chromatography (mAb-G2, lane 5; 54.1 kDa (H), 29.8 kDa (L)), and A2 after IEX column chromatography (mAb-A2, lane 6; 55.1 kDa (H), 29.8 kDa (L)). MW was calculated from the band mobilities with regards to molecular markers. b) MALDI-TOF MS spectra of glycopeptides from mAb-M3, mAb-G0, mAb-G2, mAb-A2, and mAb in the positive mode. * indicates the de-sialylated fragment ion peak. x represents the common contaminant peaks, not glycopeptides.
Fig 5.
Binding activity for FcγRIIIa of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) using the FcγRIIIa-V158-binding ELISA method.
Fig 6.
ADCC reporter gene assay of the glycoengineered anti-Her2 mAbs (mAb-M3; red square, mAb-G0; green triangle, mAb-G2; blue square, mAb-A2; purple circle), aglycosylated anti-Her2 mAb (mAb-PNGF; open diamond), fully glycosylated anti-Her2 mAb from silkworm cocoon (mAb; open square), and anti-Her2 mAb from CHO cells (trastuzumab; open circle) in SKBR-3 (a) and BT474 (b) target cells.