－ G2-peptide: Glycopeptide possessing βGal-terminal at non-reducing end of N-glycan －
It has been known that the effector function of antibody drugs closely correlates the glycan structure of Fc region. And the several information of structural relationship between Fc glycan structure and Fc receptor was accumulated recent years. Since there are some reports that not only lacking corefucose of N-glycan on Fc region but also β-galactose residues exposed at the non-reducing end shows significant effect in ADCC and CDC activities of antibody drugs,1)-3) the G2 N-glycan is being focused.
With glycan remodeling strategy by Endo-M, we attempted conversion of glycosylation on Fc region of IgG into the specific glycan that shows high effector function, and succeeded to transfer homogenous G2 N-glycans to antibodies lacking corefucose residue using G2-peptide and Glycosynthase.
A. Verification of the heavy chain size by SDS-PAGE
From comparison between the Endo-M-treated antibody and the resulting antibody with the G2 N-glycan, a band shift of the H chain of the antibody was observed while a band shift of L chain was not.
B. Detecting incorporation of G2 type glycan into Endo-M-treated antibody by lectin-blotting
In this study, ECA (Erythrina cristagalli agglutinin) was used. A band of H chain of antibody coupled with homogenous glycan was significantly stained.
C. MALDI TOF/MS analysis (Intact)
With analysis between the antibody treated by Endo-M (leaving an innermost GlcNAc residue) and the resulting antibody transferred by Glycosynthase using G2-peptide, the transfer of the homogenous G2 type glycan was evaluated by Mass spectrometry.
- 1) Assessing the Heterogeneity of the Fc-Glycan of a Therapeutic Antibody Using an engineered FcγReceptor IIIa-Immobilized Column
- 2) The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgG1 Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity
- 3) Conformational effects of N-glycan core fucosylation of immunoglobulin G Fc region on its interaction with Fcγ receptor IIIa
Related Glycoscience Topics Pages
- Oligosaccharide Replacement of a Therapeutic Antibody by using Endo-M and Glycosynthase
- Sialylglycopeptide (SGP), Disialyloctasaccharide