The interplay of protein engineering and glycoengineering to fine‐tune antibody glycosylation and its impact on effector functions

Abstract

The N‐glycan pattern of an IgG antibody, attached at a conserved site within the fragment crystallizable (Fc) region, is a critical antibody quality attribute whose structural variability can also impact antibody function. For tailoring the Fc glycoprofile, glycoengineering in cell lines as well as Fc amino acid mutations have been applied. Multiple glycoengineered Chinese hamster ovary cell lines were generated, including defucosylated (FUT8KO), α‐2,6‐sialylated (ST6KI), and defucosylated α‐2,6‐sialylated (FUT8KOST6KI), expressing either a wild‐type anti‐CD20 IgG (WT) or phenylalanine to alanine (F241A) mutant. Matrix‐assisted laser desorption ionization‐time of flight mass spectrometry characterization of antibody N‐glycans revealed that the F241A mutation significantly increased galactosylation and sialylation content and glycan branching. Furthermore, overexpression of recombinant human α‐2,6‐sialyltransferase resulted in a predominance of α‐2,6‐sialylation rather than α‐2,3‐sialylation for both WT and heavily sialylated F241A antibody N‐glycans. Interestingly, knocking out α‐1,6‐fucosyltransferase (FUT8KO), which removed core fucose, lowered the content of N‐glycans with terminal Gal and increased levels of terminal GlcNAc and Man5 groups on WT antibody. Further complement‐dependent cytotoxicity (CDC) analysis revealed that, regardless of the production cells, WT antibody samples have higher cytotoxic CDC activity with more exposed Gal residues compared to their individual F241A mutants. However, the FUT8KO WT antibody, with a large fraction of bi‐GlcNAc structures (G0), displayed the lowest CDC activity of all WT antibody samples. Furthermore, for the F241A mutants, a higher CDC activity was observed for α‐2,6‐ compared to α‐2,3‐sialylation. Antibody‐dependent cellular cytotoxicity (ADCC) analysis revealed that the defucosylated WT and F241A mutants showed enhanced in vitro ADCC performance compared to their fucosylated counterparts, with the defucosylated WT antibodies displaying the highest overall ADCC activity, regardless of sialic acid substitution. Moreover, the FcγRIIIA receptor binding by antibodies did not always correspond directly with ADCC result. This study demonstrates that glycoengineering and protein engineering can both promote and inhibit antibody effector functions and represent practical approaches for varying glycan composition and functionalities during antibody development.

Comprehensive N- and O-Glycoproteomic Analysis of Multiple Chinese Hamster Ovary Host Cell Lines

“…The extracted glycans were subjected to Carbocolumn N-glycan clean-up and stored at 4 °C for Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis. The detailed procedure for Carbocolumn N-glycan clean-up was provided in our previous publication . The de- N -glycosylated protein-bead mixture was subjected to β-elimination to release O-glycans, followed by permethylation, which were described in detail in our previous work .…”

“…The detailed procedure for Carbocolumn Nglycan clean-up was provided in our previous publication. 44 The de-N-glycosylated protein-bead mixture was subjected to β-elimination to release O-glycans, followed by permethylation, which were described in detail in our previous work. 21 The purified glycan was analyzed using a Bruker AutoFlex MALDI-TOF spectrometer in positive reflection ion mode.…”

Role of Fc Core Fucosylation in the Effector Function of IgG1 Antibodies

“…IgG N-glycan biosynthesis starts in the endoplasmic reticulum with the addition of a pyrophosphate-dolichol precursor (Dol-P, Glc3Man9GlcNAc2) to the Asn 297 Nglycosylation site of IgG. This structure is then trimmed by glucosidases and mannosidases, as the process moves to the Golgi, leading to the formation of high mannose, hybrid and the complex types N-glycans (26,(30)(31)(32)(33). The main enzymatic reactions taking place in the Golgi are summarized in Figure 2.…”

“…Also, different production cell lines and systems may lead to antibodies with different N-glycan profiles, not only regarding fucose residues. These may in turn affect function of antibodies since carbohydrates other than fucose, for example galactose or sialic acid may affect CDC or inflammation, respectively, among others ( 33 , 53 ). A more detailed description of the role of galactosylation and sialic acid is beyond the scope of this review.…”

An engineered immunomodulatory IgG1 Fc suppresses autoimmune inflammation through pathways shared with i.v. immunoglobulin

“…The core glycan structure consists of N -acetylglucosamine (GlcNAc) and mannose residues, and may be modified by the addition of fucose, GlcNAc, galactose, and sialic acid ( Figure 1B ). Indeed, much interest in IgG glycosylation has stemmed from studies that demonstrated the effects of variable glycosylation on Fc function, including afucosylated IgG gaining enhanced binding to FcγRIIIA and more potently triggering antibody-dependent cytotoxicity in vivo than fucosylated IgG ( 21 – 25 ). Additionally, work from our group and others has shown that terminal sialylation of the IgG Fc glycan conveys antiinflammatory activity ( 26 , 27 ), and notably, approximately 10% of glycans in IVIG are terminally sialylated ( 1 , 26 ).…”