Crystallizable Fragment Glycoengineering for Therapeutic Antibodies Development

Abstract

Monoclonal antibody (mAb)-based therapeutics are the fastest growing class of human pharmaceuticals. They are typically IgG1 molecules with N-glycans attached to the N297 residue on crystallizable fragment (Fc). Different Fc glycoforms impact their effector function, pharmacokinetics, stability, aggregation, safety, and immunogenicity. Fc glycoforms affect mAbs effector functions including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) by modulating the Fc-FcγRs and Fc-C1q interactions. While the terminal galactose enhances CDC activity, the fucose significantly decreases ADCC. Defucosylated immunoglobulin Gs (IgGs) are thus highly pursued as next-generation therapeutic mAbs with potent ADCC at reduced doses. A plethora of cell glycoengineering and chemoenzymatic glycoengineering strategies is emerging to produce IgGs with homogenous glycoforms especially without core fucose. The chemoenzymatic glycosylation remodeling also offers useful avenues for site-specific conjugations of small molecule drugs onto mAbs. Herein, we review the current progress of IgG-Fc glycoengineering. We begin with the discussion of the structures of IgG N-glycans and biosynthesis followed by reviewing the impact of IgG glycoforms on antibody effector functions and the current Fc glycoengineering strategies with emphasis on Fc defucosylation. Furthermore, we briefly discuss two novel therapeutic mAbs formats: aglycosylated mAbs and Fc glycan specific antibody-drug conjugates (ADCs). The advances in the understanding of Fc glycobiology and development of novel glycoengineering technologies have facilitated the generation of therapeutic mAbs with homogenous glycoforms and improved therapeutic efficacy.

Keywords: aglycosylated monoclonal antibodies; antibody–drug conjugate; chemoenzymatic glycosylation remodeling; crystallizable fragment glycoengineering; crystallizable fragment glycosylation; effector function; homogenous glycoforms; monoclonal antibodies.

Figure 1

The structures of immunoglobulin G (IgG) and N-glycans. (A) Cartoon representations of a full-length IgG showing the functional domains. An IgG consists of two heavy chains (blue) and two light chains (red). The N-glycans are presented by the green color. Crystallizable fragment (Fc) is a dimer of C_H2, C_H3, and glycans. Antigen-binding fragment (Fab) is composed of variable heavy and light domains, as well as two constant domains (CH1 and CL). (B) The schematic structures of the possible biantennary oligosaccharides attached to human IgG-Fc at N297. The core heptasaccharide (G0) is linked in black lines; the outer arm sugar residues are attached to the core by the red dash line.

Figure 2

Glycan biosynthesis through the endoplasmic reticulum (ER) and Golgi glycosylation pathway. The biosynthesis begins with the processing of the initial high mannose N-glycan in the ER followed by transferring into the cis-Golgi to generate the core N-glycan substrate used for further diversification in the trans-Golgi. The potential glycoforms include the high mannose, hybrid, and complex structure.

Figure 3

One X-ray crystal structure of N-glycan attached to N297 of crystallizable fragment (Fc) (PDB ID: 4CDH). (A) Cartoon representation of C_H2 domain with N-glycans of biantennary complex structures. The sugar residues are represented as sticks and spheres models by PyMOL. Some non-covalent interactions between oligosaccharides and proteins are presented. (B) The structural orientations of N-glycans in Fc. The two glycans from each Fc pack against each other on the α-1,3 arms.

Figure 4

The crystal structures of non-fucosylated crystallizable fragment (Fc) (PDB ID: 3SGK) and fucosylated Fc (PDB ID: 3SGJ) complexed with FcγRIIIa with a high mannose glycan on N162. (A) Cartoon representation of non-fucosylated Fc–FcγRIIIa complex produced by PyMOL. The oligosaccharides and part of hydrogen bonding formation residues are shown in sphere and stick representation. The hydrogen bonds are depicted as black dash lines. (B) Cartoon representation of fucosylated Fc–FcγRIIIa complex. The core fucose locating at the interface of Fc N297 glycan and FcγRIIIa N162 glycans is highlighted with the dot representation.

Figure 5

Representative crystal structures of IgG1 G0F, and G2F glycoforms. (A) The crystal structure of G0F. N-glycans and amino acids involving hydrogen bond formation are depicted by stick and sphere models. C_H2 domain is represented as gray cartoon. The blue, green, and red colors represent GlcNAc, Man, and Fuc, respectively. Hydrogen bonds are drawn by black dash lines. (B) The crystal structure of G2F. The purple stick represents Gal, which engages several hydrogen bonds with nearby polar amino acids.

Figure 6

Use of the endoplasmic reticulum α-mannosidase inhibitors, kifunensine, to produce the high mannose glycoform with low fucose. (A,B) Deconvoluted mass spectra for heavy chains of 4Dm2m produced in the culture medium without (A) or with addition of kifunensine (B). 4Dm2m and 4Dm2m-F were treated in buffer (7.5 M guanidine–HCl, 0.1 M Tris–HCl, and 1 mM EDTA) in the presence of 20 mM DTT and incubated at 70°C for 15 min. Mass spectrometry data were acquired on an Agilent 6520 Accurate-Mass Q-TOF LC/MS System. (C) Binding affinity to FcγRIIIa measured by surface plasmon resonance on a Biacore X100 (GE Healthcare) using a single-cycle approach. (D) Comparisons of antibody-dependent cell-mediated cytotoxicity (ADCC) activity of 4Dm2m and 4Dm2m-F by using the Promega ADCC reporter assay. Jurkat T cells engineered to express human FcγRIIIa and luciferase, through which ADCC signals were monitored.

Figure 7

Knockout of GDP-fucose transporter gene in CHO cell line to generate CHO-F6 cell line for the production of afucosylated immunoglobulin Gs. (A) Deconvoluted mass spectra for heavy chain of m860 produced in the wide-type CHO cells (110). (B) Deconvoluted mass spectra for heavy chain of m860 produced in the CHO-F6 cell line. M860 and m860-F were treated in buffer (7.5 M guanidine–HCl, 0.1 M Tris–HCl, and 1 mM EDTA) in the presence of 20 mM DTT and incubated at 70°C for 15 min. Mass spectrometry data were acquired on an Agilent 6520 Accurate-Mass Q-TOF LC/MS System.