Antibody glycosylation is the covalent attachment of carbohydrate (glycan) molecules to the protein chains of antibodies. This is a common type of post-translational modification that occurs within the cell's endoplasmic reticulum and Golgi apparatus.1
Glycosylation in the Fc (fragment crystallizable) region is a key regulator of humoral immune activity, and plays crucial roles in effector function, stability, and interactions. It is also involved in complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) functions by modulation of Fcγ receptor binding.
For therapeutic antibodies, glycosylation can impact their efficacy and safety, as specific glycoforms may be required for successful therapeutic efficacy. These glycoforms can be affected by glycosylation engineering or cell culture conditions.
Glycosylation's Role in Immune Function
Glycosylation within the Fc region is a critical modulator of the antibody's interaction with immune system components. The Fc region binds to Fc gamma receptors (FcγRs) on immune cells, which triggers various immune responses. Glycan structures attached to the Fc region can influence these interactions, altering how effectively the antibody engages with FcγRs, and thereby affecting processes like:
Complement-Dependent Cytotoxicity (CDC): In this pathway, antibodies trigger the activation of the complement system, leading to the destruction of targeted cells, such as infected or cancerous cells. Proper glycosylation in the Fc region is essential for efficient complement activation.
Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC): ADCC is a mechanism where immune cells, particularly natural killer (NK) cells, recognize and kill antibody-coated target cells. The engagement of Fcγ receptors on NK cells depends on the glycosylation state of the antibody, with certain glycan profiles improving the antibody's ability to activate these immune cells.
Types of Glycosylation and Their Impact on Antibody Function
The carbohydrate structures attached during glycosylation can vary greatly, leading to different "glycoforms" of an antibody. These glycoforms can dramatically influence how the antibody behaves in terms of stability, efficacy, and immune engagement.2
Core Fucosylation: The addition of fucose to the core glycan structure is common in many antibodies. However, defucosylation (the removal of fucose) has been shown to increase binding affinity to FcγRIIIa, a receptor found on NK cells, which enhances ADCC activity. This characteristic has been exploited in therapeutic antibody design to improve the efficacy of treatments like cancer immunotherapy.
Related: CHOK1-FUT8 Afucosylated Antibody Expression
Sialylation: Sialic acid residues attached to the glycan structures can have anti-inflammatory effects. Sialylated antibodies may exhibit reduced immune activation, making them useful in certain therapeutic contexts where dampening the immune response is desirable, such as in autoimmune diseases.3
Galactosylation: The addition of galactose to glycans can enhance complement activation through the classical pathway, promoting CDC. The level of galactosylation in an antibody can influence its ability to initiate complement cascades and thus its potency in eliminating target cells.4
Antibody Glycosylation in Therapeutics
For therapeutic antibodies, glycosylation is a key determinant of both efficacy and safety. Therapeutic antibodies used to treat diseases such as cancer, autoimmune disorders, and infectious diseases rely on specific glycosylation patterns to mediate their interactions with immune cells and complement proteins.
One of the major challenges in antibody therapeutics is ensuring the correct glycoform is produced during the manufacturing process. Since antibodies are typically produced in mammalian cell lines such as Chinese hamster ovary (CHO) cells, the glycosylation profile can vary depending on the cell line, culture conditions, and bioprocessing steps. Even minor variations in the glycan structure can impact the antibody’s performance in the body.
Glycosylation Engineering: To address the need for specific glycan structures, glycosylation engineering techniques have been developed. This involves modifying the host cell’s glycosylation pathways to produce antibodies with a defined glycoform. For instance, antibodies can be engineered to lack fucose residues (afucosylated antibodies) to increase ADCC activity, making them more effective in certain cancer therapies.
Cell Culture Conditions: The conditions under which therapeutic antibodies are produced can also influence their glycosylation. Factors such as nutrient availability, temperature, and pH in the culture environment affect the activity of glycosylation enzymes. By optimizing these conditions, manufacturers can steer the glycosylation patterns towards desired profiles, enhancing the therapeutic performance of the antibodies.
Impact of Glycosylation on Antibody Stability and Half-Life
In addition to immune modulation, glycosylation affects the overall stability and half-life of antibodies in circulation. Properly glycosylated antibodies are generally more stable, both structurally and thermally, making them less prone to aggregation. Aggregation is a significant issue in therapeutic antibody production, as it can lead to reduced efficacy and increased immunogenicity, where the patient’s immune system generates antibodies against the therapeutic product.
Glycans also influence how long an antibody remains in the bloodstream, known as its serum half-life. Antibodies with longer half-lives require less frequent dosing, improving patient compliance and reducing treatment costs. Certain glycosylation patterns, such as increased sialylation, can extend the half-life of antibodies by preventing them from being rapidly cleared from the circulation.
Zheng, K., Bantog, C., & Bayer, R. (2011). The impact of glycosylation on monoclonal antibody conformation and stability. MAbs, 3(6), 568-576. https://doi.org/10.4161/mabs.3.6.17922
Irvine, E. B., & Alter, G. (2020). Understanding the role of antibody glycosylation through the lens of severe viral and bacterial diseases. Glycobiology, 30(4), 241-253. https://doi.org/10.1093/glycob/cwaa018
Bartsch, Y. C., Rahmöller, J., M. Mertes, M. M., Eiglmeier, S., M. Lorenz, F. K., Stoehr, A. D., Braumann, D., Lorenz, A. K., Winkler, A., Lilienthal, M., Petry, J., Hobusch, J., Steinhaus, M., Hess, C., Holecska, V., Schoen, C. T., Oefner, C. M., Leliavski, A., Blanchard, V., . . . Ehlers, M. (2018). Sialylated Autoantigen-Reactive IgG Antibodies Attenuate Disease Development in Autoimmune Mouse Models of Lupus Nephritis and Rheumatoid Arthritis. Frontiers in Immunology, 9. https://doi.org/10.3389/fimmu.2018.01183
Peschke, B., Keller, C. W., Weber, P., Quast, I., & Lünemann, J. D. (2017). Fc-Galactosylation of Human Immunoglobulin Gamma Isotypes Improves C1q Binding and Enhances Complement-Dependent Cytotoxicity. Frontiers in Immunology, 8. https://doi.org/10.3389/fimmu.2017.00646
Antibody specificity refers to an antibody's ability to selectively bind to a unique epitope on a target antigen while avoiding interactions with unrelated antigens. This property arises from the highly specialized antigen-binding site located in the variable region of the antibody, which determines its unique binding characteristics.
Antibody affinity refers to the strength of the binding interaction between a single antigen epitope and the paratope (binding site) of an antibody. This interaction is a fundamental measure of how well an antibody recognizes its specific antigen target.
Recombinant antibodies are produced using genetic engineering techniques, unlike traditional antibody production, where the immune system generates antibodies without direct control over their sequence. By introducing genes encoding antibody fragments into host cells, such as bacteria or mammalian cells, recombinant antibodies can be expressed, purified, and deployed for applications including research, diagnostics, and therapeutics.
Recombinant antibody expression is a biotechnological process that involves engineering and producing antibodies outside their natural context using recombinant DNA technology.
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