How to Enhance ADCC

Why Enhance ADCC

Antibodies are composed of antigen-binding sites (Fab) and crystallizable sites (Fc). The Fab region binds to specific antigens, thus determining the specificity and affinity of the antibody, and the Fc region binds to Fc receptors (FcγRI, FcγRII, FcγRIII) expressed on the surface of immune cells, complement (C1q) and FcRn in the blood, thereby activating immune effector cells to clear foreign substances.

The structure of an antibody determines its mechanism of action, with the Fab region recognizing free molecules and cell surface receptors to determine the specificity and affinity of antibody recognition, while the Fc region determines the effector function of the antibody, including antibody-dependent cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).¹

Fc receptors have the potential to influence the efficacy of anti-cancer antibodies, and when they bind to the Fc region of antibodies, they can induce ADCC and ADCP. Different immune cells express specific Fc receptors, e.g., neutrophils usually express FcγRI, FcγRII, and FcγRIIIb, while NK cells only express FcγRIIIa. FcγRIIIa is generally considered to be a key receptor for ADCC, so while NK cells, monocytes, macrophages, and neutrophils can all produce ADCC effects, NK cells are considered to be the most important cell population.

Fc Glycosylation Modification

The glycosylation of the N297 position of the antibody heavy chain plays an important role in the Fc effector function, and the glycosylation at this site can affect the binding of the Fc receptor and C1q to Fc, thereby affecting the corresponding cellular effect. In IgG antibodies, the glycosylated core of N297 is mainly composed of N-GlcNAc and other sugars, among which fucosylated GlcNAc is the most common, and the presence of fucose will hinder the binding of Fc to the receptor FcγRIIIa through steric hindrance, thereby attenuating the ADCC effect. Therefore, the removal of fucose can enhance the binding of FcγRIIIa to Fc and enhance the ADCC effect. You can learn more about Glycosylation Engineering technology at Creative Biolabs by clicking here.

Another method of engineering to reduce antibody fucose levels is to knock out the FUT8 gene in host cells by means of genetic engineering. Within mammalian cells, fucose binds to glucosamine on the Fc terminus of the antibody via the α-1,6 glycosidic bond. This glycosidic bond is catalyzed by the α-1,6-fucosyltransferase encoded by the FUT8 gene. Knockdown of the FUT8 gene can eliminate fucose at the Fc terminus of the antibody, thereby increasing the ADCC effect of the antibody. Creative Biolabs' Afuco ^TM technology platform is an ADCC-enhanced platform built by knocking out the FUT8 gene. Subsequently, through further detailed research on fucose modification, it was found that a variety of enzymes were involved in this process, and a series of antibody expression engineering cell lines were born through the knockout of these enzymes.

Site-Directed Mutations

Amino acid mutations are introduced in the relevant regions of Fc to increase ADCC and ADCP effects. For example, a combination of S298A/E333A/K334A mutations in IgG and IgE has been shown to enhance the binding of Fc to FcγRIIIa while weakening the binding to FcγRIIb.

In addition, structure-based computational models combined with high-throughput screening can identify amino acid mutations that can enhance Fc effects. For example, anti-CD52 antibodies, when mutated in S239D/I332E or S239D/A330L/I332E, were found to have increased binding affinity to both FcγRIIIa and FcγRIIb. The study found that these two mutations greatly increased the ADCC effect, and the ADCP effect was also slightly improved.

G236A/S239D/A330L/I332E or G236A/A330L/I332E mutations can also significantly enhance the affinity of Fc for FcγRIIa and FcγRIIIa, while the latter binds to the inhibitory receptor FcγRIIb relatively weakly.

The F243L/R292P/Y300L/V305I/P396L mutation is an amino acid mutation obtained by yeast display technology, which enhances the affinity of Fc to FcγRIIIa and reduces the binding capacity to FcγRIIb, thereby enhancing the Fc effector function.

Site-directed mutagenesis has become one of the important means to improve the effect of antibody ADCC. Creative Biolabs has been at the forefront of developments, specializing in providing our customers with site-directed mutagenesis technology to obtain ADCC/CDC-enhanced antibodies. You can learn more about this by clicking here.

Fc Region Multimerization

Compared with glycosylation and site-directed mutagenesis, this method enhances the ADCC effect by increasing the number of Fc regions of IgG1, so that one IgG1 carries multiple Fc regions and binds multiple FcγRIIIa. This is a more novel and straightforward approach.

If you are interested in the ADCC/CDC/ADCP program, welcome to contact us.