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.

Considerations for the Design of Antibody-Based Therapeutics

“…These complex N-glycans contain a core heptasaccharide motif with 4 N-acetylglucosamine (GlcNAc) residues and 3 mannose residues in a biantennary arrangement. 8,149 Fucose may be added to the protein-proximal GlcNAc residue, bisecting GlcNAc may be added to the central mannose residue, galactose may be added to each terminal mannose residue, and sialic acid (N-acetylneuraminic acid in mammals or N-glycolylneuraminic acid in some nonhuman mammals) may be additionally added to these galactose residues. All these possibilities for individual glycans, combined with the potential for differential glycosylation on each heavy chain, allow for significant heterogeneity in otherwise similar molecules of antibody.…”

“…Common strategies to modify glycosylation in cell culture include the addition of glycan precursors to increase saccharide incorporation or glycosyltransferase inhibitors to decrease incorporation. 149,159 In addition, the genes for glycosylating enzymes within host cells may be knocked out to reduce fucosylation and nonhuman glycosylation or knocked in to provide a more human-like glycosylation profile. Clinical trials such as one with the Food and Drug Administrationeapproved obinutuzumab have demonstrated that low-fucose glycovariants from engineered cell lines may lower the risk of disease progression, but increase adverse events, compared with nonglycoengineered antibodies.…”

Boosting therapeutic potency of antibodies by taming Fc domain functions

“…For example, margetuximab, developed by Mac-roGenics, had adapted the same Fab target (Her2) as transtuzumab but had been Fc-engineered to maximize immune effector function by elevating relative affinity to activating Fcγ receptor, FcγRIIIa over inhibitory Fcγ receptor, FcγRIIb, and it recently showed a 24% risk reduction in patients relative to that of trastuzumab in a phase 3 clinical trial in 536 breast cancer patients 69 . Other significant efforts have been directed to the engineering of antibodies with improved affinity for FcγRIIIa and enhanced effector function by amino acid mutations 36,[70][71][72][73][74] or glycan modifications [75][76][77][78][79][80][81] . Hatori and coworkers also engineered an antibody Fc variant (P238D/L328E) that showed selectively enhanced FcγRIIb binding over both FcγRIIa-R131 and FcγRIIa-H131 82 .…”

Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells

“…Gramer et al (2011) galactosylated species and, therefore, confirmed the possibility of obtaining high galactosylation levels in vitro. This may be overcome by protein engineering in the Fc region Li, Zhu, Chen, Feng, & Dimitrov, 2017). In addition, because Fc-glycans are rather protected in the Fc part of the antibody, enzymes might be sterically hindered from interacting with the glycans (Nigrovic, 2013).…”

“…In addition, because Fc-glycans are rather protected in the Fc part of the antibody, enzymes might be sterically hindered from interacting with the glycans (Nigrovic, 2013). This may be overcome by protein engineering in the Fc region Li, Zhu, Chen, Feng, & Dimitrov, 2017).…”