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.

Keywords: Therapeutic monoclonal antibody; antibody-dependent cell-mediated cytotoxicity; complement-dependent cytotoxicity; galactose; glycoengineering.

Figure 1.

Major glycan structures of therapeutic mAbs. (a) Core-fucosylated agalacto-biantennary complex-type glycan (G0F); (b) core-fucosylated biantennary complex-type glycan with galactosylation on the Man α1-6 arm (G1aF); (c) core-fucosylated biantennary complex-type glycan with galactosylation on the Man α1-3 arm (G1bF); (d) core-fucosylated and fully galactosylated biantennary complex-type glycan (G2F). Yellow circle, Gal; green circle, Man; blue square, N-acetylglucosamine; red triangle, fucose.

Figure 2.

Preparation of anti-CD20 mAbs with core-fucosylated homogeneous N-glycans. This illustration details the preparation of anti-CD20 mAbs having two N-glycan chains with G2F structure. (1) digestion with wild-type Endo S and Endo D for truncating N-glycans, (2) transglycosylation with Endo F3 mutant (D165Q) in the presence of oxazolinated glycans as a glycan donor, (3) the separation and purification of anti-CD20 mAbs with two core-fucosylated N-glycan chains using cation-exchange column chromatography. Yellow circle, Gal; green circle, Man; blue square, N-acetylglucosamine; red triangle, fucose.

Figure 3.

Glycan profiles of mAbs with homogeneous glycans. Glycan profiles of commercially available anti-CD20 mAb (a) and glycoengineered G0F mAb (b), G1aF mAb (c), G1bF mAb (d), and G2F mAb (e). The glycan profiles were obtained using HPLC analysis of 2-AB-labeled glycans.

Figure 4.

CDC and C1q-binding activities of glycoengineered anti-CD20 mAbs. (a, b) CDC activities of glycoengineered anti-CD20 mAbs. Raji cells were incubated with 16% human serum and were serially diluted with glycoengineered anti-CD20 mAbs. (a) Percentages of cell lysis plotted against mAb concentrations. (b) CDC activity of 1 µg/ml of G1aF mAb and G1bF mAb mixtures in different ratios. (c) C1q binding of glycoengineered anti-CD20 mAbs. Raji cells were opsonized with anti-CD20 mAbs and incubated with human serum. The cells were stained with FITC-conjugated anti-C1q antibody and the C1q-binding level was analyzed by flow cytometer. Data are presented as mean ± SD (n = 3).

Figure 5.

Binding of glycoengineered anti-CD20 mAbs to human FcγRs. SPR analysis was used to measure the binding of anti-CD20 mAbs to human FcγRI, FcγRIIa, and FcγRIIIa. Binding sensorgrams corrected by both the surface blank and buffer injection control are represented.

Figure 6.

FcγR activation properties of glycoengineered anti-CD20 mAbs. FcγRIIa (a) and FcγRIIIa (b) activation properties of glycoengineered mAbs. Jurkat/FcγRIIa/NFAT-Luc or Jurkat/FcγRIIIa/NFAT-Luc reporter cells were incubated with serially diluted anti-CD20 mAbs in the presence of Raji cells. FcγR activation was evaluated by assessing the luminescence intensity.

Figure 7.

Comparison of structural stabilities of the CH2 domain of glycoengineered anti-CD20 mAbs using HDX/MS. Deuterium uptake plots of peptides Phe245–Leu255 (FLFPPKPKDTL) (a), Leu246–Leu255 (LFPPKPKDTL) (b), and Leu246–Met256 (LFPPKPKDTLM) (c) in the H-chain. (d) Physical representations of the crystal structures (PDB 1HZH) of peptides at Phe245–Met256 (magenta ribbons).