Afucosylation is an advanced glycoengineering technique for therapeutic antibodies to optimize immune responses, particularly antibody-dependent cellular cytotoxicity (ADCC). By removing fucose from the core region of antibody glycans, afucosylation improves the binding affinity between antibodies and FcγRIIIa receptors on immune cells, such as natural killer (NK) cells. This increased affinity strengthens ADCC, making afucosylated antibodies highly effective for treating cancers and other diseases.
Importance of ADCC in Monoclonal Antibody Therapy
ADCC is a critical immune response that drives the efficacy of many IgG1 mAbs by bridging immune effector cells, like NK cells, to tumor cells coated with antibodies. Once the antibody binds to a tumor antigen, its Fc region interacts with FcγRIIIa receptors on NK cells, initiating cytotoxic responses that lead to the target cell’s death. For many cancer therapies, including those using rituximab and trastuzumab, ADCC is the principal mechanism of action. Enhancing this process through afucosylation can significantly improve therapeutic potency by strengthening the Fc-FcγRIIIa interaction, thus enabling higher ADCC levels.
Afucosylation involves the removal of fucose from the glycan at the N297 position within the antibody's Fc region. Fucose is a sugar molecule attached by an enzyme called FUT8, and its absence in the Fc glycan enhances ADCC by increasing FcγRIIIa binding affinity. Afucosylated antibodies display 10- to 50-fold greater binding affinity to FcγRIIIa compared to their fucosylated counterparts, resulting in significantly improved NK cell-mediated ADCC. This effect is particularly prominent when FcγRIIIa is the high-affinity variant V158, which naturally binds IgG1 more strongly than the F158 variant.
There are developed several methods to produce afucosylated antibodies, including:
Glycoengineering in Cell Lines: Many afucosylated antibodies are produced in genetically modified cell lines, like Chinese hamster ovary (CHO) cells, which lack FUT8 and therefore produce antibodies with minimal core fucosylation.
Enzymatic Modification: Techniques using glycosyltransferases enable site-specific removal of fucose, creating uniform afucosylated antibodies.
Chemical Synthesis: Synthetic methods allow precise control over glycosylation, though currently less common due to complexity and cost.
The improved binding between afucosylated antibodies and FcγRIIIa receptors is largely driven by structural factors. In fucosylated antibodies, the core fucose creates steric hindrance that reduces the FcγRIIIa's binding affinity by preventing close contact between the antibody's Fc region and the receptor’s Asn162 glycan. Removing fucose eliminates this hindrance, allowing more stable hydrogen bonds and van der Waals forces between the Fc and FcγRIIIa. X-ray crystallography studies reveal that the enhanced binding arises from newly formed carbohydrate-carbohydrate interactions that strengthen receptor binding, particularly when the FcγRIIIa receptor itself is glycosylated at Asn162.
In practical terms, the absence of fucose on one or both Fc chains of the antibody can increase FcγRIIIa binding by as much as 20- to 30-fold, significantly amplifying the antibody's ADCC potential.
Besides afucosylation, other modifications, such as increased galactosylation and sialylation, influence ADCC, albeit less significantly. Galactosylation, the addition of galactose residues, can slightly improve FcγRIIIa binding, while sialylation can reduce ADCC if fucose is present but has a negligible effect in afucosylated antibodies.
Clinical Impact of Afucosylated Antibodies
Afucosylated antibodies have shown marked success in clinical trials, often with improved outcomes in patients with cancer. For instance:
Anti-CD20 Antibodies: In colorectal cancer patients treated with anti-CD20 antibodies, individuals with the FcγRIIIa-V158 variant responded particularly well to afucosylated forms of the drug due to the increased FcγRIIIa binding.
Anti-HER2 Antibodies: Afucosylated anti-HER2 antibodies targeting breast cancer cells also demonstrated improved ADCC, particularly benefiting patients whose immune cells carried high-affinity FcγRIIIa variants.1
References:
Pereira, N. A., Chan, K. F., Lin, P. C., & Song, Z. (2018). The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity. MAbs, 10(5), 693–711. https://doi.org/10.1080/19420862.2018.1466767
