Glycoengineering of human IgG1-Fc through combined yeast expression and in vitro chemoenzymatic glycosylation

Abstract

The presence and precise structures of the glycans attached at the Fc domain of monoclonal antibodies play an important role in determining antibodies' effector functions such as antibody-dependent cell cytotoxicity (ADCC), complement activation, and anti-inflammatory activity. This paper describes a novel approach for glycoengineering of human IgG1-Fc that combines high-yield expression of human IgG1-Fc in yeast and subsequent in vitro enzymatic glycosylation, using the endoglycosidase-catalyzed transglycosylation as the key reaction. Human IgG1-Fc was first overproduced in Pichia pastoris. Then the heterogeneous yeast glycans were removed by Endo-H treatment to give the GlcNAc-containing IgG1-Fc as a homodimer. Finally, selected homogeneous glycans were attached to the GlcNAc-primer in the IgG1-Fc through an endoglycosidase-catalyzed transglycosylation, using sugar oxazolines as the donor substrates. It was found that the enzymatic transglycosylation was efficient with native GlcNAc-containing IgG1-Fc homodimer without the need to denature the protein, and the reaction could proceed to completion to give homogeneous glycoforms of IgG1-Fc when an excess of oligosaccharide oxazolines was used as the donor substrates. The binding of the synthetic IgG1-Fc glycoforms to the FcgammaIIIa receptor was also investigated. This novel glycoengineering approach should be useful for providing various homogeneous, natural or synthetic glycoforms of IgG1-Fc for structure-function relationship studies, and for future clinical applications.

Figure 1. Schematic presentations of human IgG type antibody and the Fc glycans

(A), structural features of human IgG antibody; (B), a hinge-containing IgG1-Fc dimer in which the trimannose core N-glycans were remodeled at the Asn-297 sites. This model was based on the crystal structure of an anti-HIV antibody b12 (PDB code, 1hzh) (E. O. Saphire, et al, Science, 2001, 293, 1155). (C), the structure of the N-glycans attached to Asn-297.

Figure 2. The map of the yeast expression vector (A) and the cDNA and the amino acid sequence of the recombinant human IgG1-Fc (B)

The N-glycosylation site was underscored; the C-myc epitope and the His-tag at the C-terminus of the recombinant protein was in bold.

Figure 3. Characterization of the yeast expressed IgG1-Fc

A, SDS-PAGE. The yeast-expressed IgG1-Fc (lanes 2 and 3) and PNGase F treated IgG1-Fc (lanes 4 and 5) were shown. SDS-PAGE were run under non-reducing conditions (lanes 2 and 4) and reducing conditions (lanes 3 and 5). Lane 1 is the protein marker. The protein bands were visualized by staining with Coomassie brilliant blue dye; B, Gel filtration chromatography of the purified IgG1-Fc and the PNGase F-treated (deglycosylated) IgG1-Fc. The size labels (158, 44, and 17 kDa) were deduced from the appearance of the standard proteins γ-globulin, ovalbumin, and myoglobin, respectively; C, the MALDI-TOF of the N-glycans released from the recombinant IgG1-Fc. The symbols M₉ to M₁₃ represent yeast glycans Man₉GlcNAc₂ to Man₁₃GlcNAc₂, respectively.

Figure 4. Schematic presentation of the glyco-engineering of human IgG1-Fc

The heterogeneous N-glycans of the yeast-expressed IgG1-Fc was removed by Endo-H treatment. Then the GlcNAc-containing IgG1-Fc was used as an acceptor for the Endo-A catalyzed transglycosylation, with Man₃GlcNAc-oxazoline and Gal₂Man₃GlcNAc-oxazoline as the donor substrates, respectively.

Figure 5. Analysis of IgG1-Fc glycoforms

A, de-glycosylation with Endo-H: lane 1, protein marker; lane 2, recombinant IgG1-Fc (1); and lane 3, GlcNAc-IgG1-Fc (2) obtained by Endo-H treatment. B, gel filtration chromatography of GlcNAc-IgG1-Fc. The size labels (158, 44, and 17 kDa) were deduced from the appearance of the standard proteins γ-globulin, ovalbumin, and myoglobin, respectively; C, analysis of transglycosylation products: lane 1, protein marker; lane 2, GlcNAc-IgG1-Fc (2); lane 3, Man₃GlcNAc₂-glycoform (4); lane 4, GlcNAc-IgG1-Fc (2); lane 5, Gal₂Man₃GlcNAc₂-glycoform (6). D, analysis of transglycosylation products by PNGase F deglycosylation: lane 1, protein marker; lane 2, glycoform (6); lane 3, deglycosylation of 6 by PNGase F; lane 4, glycoform (4); lane 5, deglycosylation of 4 by PNGase F.

Figure 6. MALDI-TOF MS analysis of glycans in the IgG1-Fc glycoforms

The glycans were released from the IgG1-Fc glycoforms by PNGase F treatment and analyzed by MALDI-TOFMS. A, glycan released from glycoform Man₃GlcNAc₂-Fc (4); B, glycan released from glycoform Gal₂Man₃GlcNAc₂-Fc (6); and C, glycans released from the IgG-Fc of Cetuximab. G0F, G1F, and G2F represent the fucosylated bi-antennary complex type N-glycans with 0, 1, and 2 terminal galactose residues, respectively.

Figure 7

SPR Sensorgrams of the binding of different IgG1-Fc glycoforms with immobilized FcγRIIIa