Antibody function is highly dependent on the glycosylation status of the Fc region. Immunoglobulin G (IgG) antibodies are Y-shaped and composed of two identical heavy chains and two identical light chains. Each heavy chain comprises three constant domains (CH1, CH2, CH3) and a hinge region, while the light chains contain one constant and one variable domain. The Fc region, consisting of the CH2 and CH3 domains of the heavy chains, mediates interactions with immune effector molecules and receptors.
A conserved N-linked glycosylation site is located at Asn297 in the CH2 domain of each heavy chain. The glycan moiety at this site plays a critical structural role, stabilizing the Fc conformation and enabling binding to Fc gamma receptors (FcγRs) and complement component C1q.
Therapeutic monoclonal antibodies (mAbs) that recognize cell-surface antigens can engage FcγRs on effector cells such as natural killer (NK) cells, monocytes, macrophages, neutrophils, eosinophils, and dendritic cells, or bind to C1q to trigger the classical complement cascade. These interactions mediate antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). Effector cell–dependent crosslinking can also promote apoptosis of target cells.
The FcγR family comprises high-affinity (FcγRI) and low-affinity (FcγRII and FcγRIII) receptors, which can be either activating (FcγRI, FcγRIIa, FcγRIIIa) or inhibitory (FcγRIIb).1
→ Fc glycans at Asn297 shape antibody effector functions, meaning that controlling this structural feature offers a path to improving therapeutic antibody performance.
The Fc glycans at Asn297 are mostly complex biantennary N-glycans in antibodies produced in mammalian cell lines. Variability in glycan composition (known as microheterogeneity) arises from differences in terminal sugar residues and core modifications. Key structural variants include:
Core α1,6-linked fucose
Terminal galactose
Terminal sialic acid
Bisecting N-acetylglucosamine
High mannose or hybrid-type glycans
The combination of these features yields multiple glycoforms, e.g., G0F, G1F, G2F for fucosylated structures, and G0, G1, G2 for nonfucosylated structures.2
Although N-glycans can also occur in the variable (Fab) domains in ~15–20% of IgG molecules, these typically differ from Fc glycans in composition and have limited influence on Fc-mediated effector functions.
The glycan profile of recombinant antibodies depends on the expression system. Chinese hamster ovary (CHO) cells typically produce human-compatible complex-type glycans, whereas murine cell lines may add non-human epitopes such as α1,3-galactose, and plant expression systems can introduce β1,2-xylose and α1,3-fucose linkages.
→ Fc glycans exhibit structural microheterogeneity which differ by expression system and influence antibody function.
Related: Afucosylated Antibodies
The Fc glycan composition directly influences the interaction between IgG and FcγRs or C1q. Variations in glycan structure can modulate receptor affinity and downstream effector functions:
Core fucose: Absence of α1,6-linked fucose increases FcγRIIIa and FcγRIIIb binding by 10–100-fold, leading to markedly enhanced ADCC.
Galactosylation: Increases C1q binding and CDC potency; can also modestly influence ADCC, especially in afucosylated antibodies.
Sialylation: Often linked to anti-inflammatory properties; reduces FcγRIIIa binding and ADCC.
Bisecting GlcNAc: Inhibits fucosylation, thereby indirectly increasing FcγRIIIa binding affinity.
High mannose: May enhance FcγRIIIa binding but generally results in faster clearance due to mannose receptor–mediated uptake.3
Structural studies have shown that core fucose affects the carbohydrate–carbohydrate interface between Fc and FcγRIIIa glycans, altering both the on-rate and stability of binding.
→ The specific composition of Fc glycans alters FcγR and C1q binding affinity, thereby modulating ADCC, ADCP, and CDC potency.
The presence or absence of core fucose at Asn297 is the single most impactful modification in determining FcγRIII binding strength. Afucosylated IgG1 exhibits:
10–100× higher affinity for FcγRIIIa/b.1
2–40× increased ADCC by NK cells, depending on galactosylation level.
Reduced inhibition of NK cell activity by circulating plasma IgG.
In addition to NK cells, FcγRIIIa-positive monocytes and macrophages can mediate increased ADCP with afucosylated antibodies, although the effect is context-dependent. FcγRIIIB-positive neutrophils also show higher binding to afucosylated IgG1, influencing trogocytosis and cytokine release.
Clinically, afucosylation has been harnessed in several approved mAbs, including obinutuzumab and mogamulizumab, to improve target cell depletion. Naturally occurring afucosylated antibodies are also observed in specific physiological and pathological contexts. During pregnancy alloimmunization and infections with certain enveloped viruses or parasites, antigen-specific IgG1 can show markedly reduced fucosylation. In some cases, such as dengue virus and SARS-CoV-2 infection, low fucosylation correlates with disease severity, possibly due to heightened FcγRIII-mediated immune activation.
→ Targeted removal of core fucose is a service provided by Biointron’s Afucosylated Antibody Expression platform
Related: Afucosylated Antibody Expression
While core fucose has the largest quantitative impact on FcγRIII affinity, other glycan features contribute to effector function modulation:
Galactosylation: Promotes CDC via improved C1q binding. May also influence anti-inflammatory signaling through cooperative FcγRIIb–dectin-1 engagement.
Sialylation: Associated with anti-inflammatory activity, as demonstrated in the context of intravenous immunoglobulin (IVIG) therapy.
Bisecting GlcNAc: Enhances FcγRIII binding indirectly by blocking fucosylation.
High mannose: Can enhance FcγRIIIa binding but generally shortens serum half-life via mannose receptor–mediated clearance.
These modifications can act additively or synergistically. For example, galactosylation of an afucosylated IgG further increases FcγRIIIa binding.
Certain Fc glycan structures can pose safety or immunogenicity risks. For example, α1,3-galactose, which can be present on monoclonal antibodies produced in murine cell lines, has been linked to hypersensitivity reactions in patients receiving cetuximab due to pre-existing IgE antibodies. N-glycolylneuraminic acid (NGNA), found in some non-human mammalian production systems, is potentially immunogenic and has been associated with the development of anti-drug antibodies. In addition, β1,2-xylose and α1,3-fucose, which occur on plant-derived glycans, can be recognized by IgE in individuals with pollen allergies, posing a possible risk of allergic reactions.
Several afucosylated therapeutic antibodies have reached the market:
Mogamulizumab (brand name: Poteligeo) is an anti-CCR4, humanized IgG1 monoclonal antibody that is afucosylated to improve FcγRIIIa binding and enhance ADCC; it was first approved in Japan in 2012 for the treatment of mycosis fungoides or Sézary syndrome.
Benralizumab (brand name: Fasenra) is an anti-IL-5Rα, humanized IgG1 monoclonal antibody that is afucosylated to improve FcγRIIIa binding and enhance ADCC; it was first approved in the United States in 2017 for the treatment of asthma.
Inebilizumab (brand name: Uplizna) is an anti-CD19, humanized IgG1 monoclonal antibody that is afucosylated to improve FcγRIIIa binding and enhance ADCC; it was first approved in the United States in 2020 for the treatment of neuromyelitis optica spectrum disorders.
Belantamab mafodotin (brand name: BLENREP) is an anti-BCMA, humanized IgG1 antibody–drug conjugate with a noncleavable maleimidocaproyl linker to the cytotoxic payload monomethyl auristatin F (MMAF), and is afucosylated to improve FcγRIIIa binding and enhance ADCC; it was first approved in the United States in 2020 for the treatment of multiple myeloma.4
Biointron offers an afucosylated antibody production service designed to enhance antibody-dependent cellular cytotoxicity (ADCC) without altering antigen specificity. Knocked out Fut8 cell line is applied in producing afucosylated antibodies. Fut8 is an enzyme that catalyzes the formation of a-1,6 fucosyl glycosidic bonds, and thus the addition of fucose to asparagine-linked N-acetylglucosamine moieties. Biointron's fucose-free host cell CHOK1BN-Fut8KO can be used for industrial production after antibody characterization and functional verification. Compared to the unmodified CHOK1BN cell line, the antibody produced by the modified cell line not only achieves afucosylation but also maintains consistency in terms of expression quantity and quality.
Golay, J., Andrea, A. E., & Cattaneo, I. (2022). Role of Fc Core Fucosylation in the Effector Function of IgG1 Antibodies. Frontiers in Immunology, 13, 929895. https://doi.org/10.3389/fimmu.2022.929895
Reusch, D., & Tejada, M. L. (2015). Fc glycans of therapeutic antibodies as critical quality attributes. Glycobiology, 25(12), 1325. https://doi.org/10.1093/glycob/cwv065
Barrientos, G., Habazin, S., Novokmet, M., Almousa, Y., Lauc, G., & Conrad, M. L. (2020). Changes in subclass-specific IgG Fc glycosylation associated with the postnatal maturation of the murine immune system. Scientific Reports, 10(1), 1-10. https://doi.org/10.1038/s41598-020-71899-7
The Antibody Society. Therapeutic monoclonal antibodies approved or in regulatory review. (date accessed); www.antibodysociety.org/antibody-therapeutics-product-data
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