Effects of terminal galactose residues in mannose α1-6 arm of Fc-glycan on the effector functions of therapeutic monoclonal antibodies

Abstract

Typical crystallizable fragment (Fc) glycans attached to the CH2 domain in therapeutic monoclonal antibodies (mAbs) are core-fucosylated and asialo-biantennary complex-type glycans, e.g., G2F (full galactosylation), G1aF (terminal galactosylation on the Man α1-6 arm), G1bF (terminal galactosylation on the Man α1-3 arm), and G0F (non-galactosylation). Terminal galactose (Gal) residues of Fc-glycans are known to influence effector functions such as antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (CDC), but the impact of the G1F isomers (G1aF and G1bF) on the effector functions has not been reported. Here, we prepared four types of glycoengineered anti-CD20 mAbs bearing homogeneous G2F, G1aF, G1bF, or G0F (G2F mAb, G1aF mAb, G1bF mAb, or G0F mAb, respectively), and evaluated their biological activities. Interestingly, G1aF mAb showed higher C1q- and FcγR-binding activities, CDC activity, and FcγR-activation property than G1bF mAb. The activities of G1aF mAb and G1bF mAb were at the same level as G2F mAb and G0F mAb, respectively. Hydrogen–deuterium exchange/mass spectrometry analysis of dynamic structures of mAbs revealed the greater involvement of the terminal Gal residue on the Man α1-6 arm in the structural stability of the CH2 domain. Considering that mAbs interact with FcγR and C1q via their hinge proximal region in the CH2 domain, the structural stabilization of the CH2 domain by the terminal Gal residue on the Man α1-6 arm of Fc-glycans may be important for the effector functions of mAbs. To our knowledge, this is the first report showing the impact of G1F isomers on the effector functions and dynamic structure of mAbs. Abbreviations: ABC, ammonium bicarbonate solution; ACN, acetonitrile; ADCC, antibody-dependent cell-mediated cytotoxicity; C1q, complement component 1q; CDC, complement-dependent cytotoxicity; CQA, critical quality attribute; Endo, endo-β- N -acetylglucosaminidase; FA, formic acid; Fc, crystallizable fragment; FcγR, Fcγ receptors; Fuc, fucose; Gal, galactose; GlcNAc, N -acetylglucosamine; GST, glutathione S-transferase; HER2, human epidermal growth factor receptor 2; HDX, hydrogen–deuterium exchange; HILIC, hydrophilic interaction liquid chromatography; HLB-SPE, hydrophilic-lipophilic balance–solid-phase extraction; HPLC, high-performance liquid chromatography; mAb, monoclonal antibody; Man, mannose; MS, mass spectrometry; PBS, phosphate-buffered saline; SGP, hen egg yolk sialylglycopeptides.

Proteoform-Resolved FcɤRIIIa Binding Assay for Fab Glycosylated Monoclonal Antibodies Achieved by Affinity Chromatography Mass Spectrometry of Fc Moieties

“…Mainly, several partially separated species in the EICs of Figure 3 indicate separation of glycoforms with terminal galactose present on the 1,3-arm or the 1,6-arm of G1F. Differential FcɤRIIIa affinity of G1F(1,3) and G1F(1,6) glycoforms has recently been reported [G2F = G1F(1,6) > G1F(1,3) = G0F] (Aoyama et al, 2019). Based on this, G0F/G1F(1,3) has a similar affinity as G0F/G0F.…”

Fc galactosylation follows consecutive reaction kinetics and enhances immunoglobulin G hexamerization for complement activation

“…The only peptide segment with a different H/D exchange uptake between hypogalactosylated and hypergalactosylated mAb ( Figure 3b ) is peptide 246–257 located in the CH2 domain, which agrees well with the previous study. 32 , 33 This specific region was found to be a critical site for hexamer formation 31 , 34 (orange cluster in Figure 3c ).…”

Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life

“…The Asn297 glycan within the CH2 domain of the Fc can be modified to improve CDC activity (56, 57, 98–101). In a large screen of 20 different glycoforms of anti-trinitrophenyl hapten IgG1 Fc an overabundance of galactosylation increased C1q binding and CDC activity compared to the unmodified glycoform of IgG1 (57).…”