Antibody-dependent cellular cytotoxicity (ADCC) is one of the main mechanisms of action of therapeutic monoclonal antibodies, which recognize and kill target cells expressing specific antigens through effector cells expressing Fc receptors, and require antibodies with Fc and Fab regions to participate.
ADCC can be mediated by a variety of effector cells, which share three common features: leukocytes, granule-containing, and FcR-expressing. Cells that meet these conditions are: monocytes (NK cells, macrophages, γδ T cells) and polymorphonuclear leukocytes (neutrophils, basophages, eosinophils), among which NK cells are the most common research objects in terms of ADCC effect.
Creative Biolabs offers a range of ADCC reporter effector cells, find your cell of interest in the ADCC Cell Line.
There are three types of FcR mediating IGg-dependent ADCC effects: FcγRI (CD64), FcγRII (CD32), and FcγRIIIA (CD16), among which FcγRIIIA (CD16) is mainly expressed by NK cells, so most experiments focus on FcγRIIIA (CD16).
Table 1. Effector cells involved in ADCC and major Fc receptors in peripheral blood. (Creative Biolabs)
|FcγRIIIA / CD16
|FcγRI / CD64, FcγRII / CD32
|γδ T cells (subset)
|FcγRIIIA / CD16
|Granulocytes (neutrophils, basophils, eosinophils)
|FcγRII / CD32C, FcγIIIb / CD16, FcαRI / CD89
A prerequisite for effector cells to be activated is that the antibody Fab region binds to the target cell. After the antibody binds to the target cells, the conformation of its Fc region changes, resulting in an increase in the FcR affinity of the antibody for effector cells.
The affinity of the antibody to FcR is affected by glycosylation or amino acids, for example, when fucose on the antibody is heavily glycosylated, the affinity of the antibody for FcγRIIIA (CD16a) will be reduced, and if fucose is removed, the affinity of the antibody for CD16a and the ability to mediate the ADCC effect will be enhanced; In rituximab-based experiments, there is a trend that the expression of amino acids in the 158-position polymorphism of CD16a as valine (V) or phenylalanine (F) affects the affinity of the antibody to effector cells, and the trend is roughly V/V>V/F>F/F.
Fig.1 The affinity of mAb for the effector cell receptor FcγRIIIA (CD16) is altered by amino acid substitution. ¹
Cellular src kinases phosphorylate immune receptor tyrosine-based activation motifs (ITAMs) in effector cells. Upon cross-linking of the antibody to CD16, the src kinase is activated and the ITAM on the CD16γ subunit is phosphorylated.
The phosphorylated ITAM recruits Syk and activates its associated signaling pathways, such as PLC-γ, PI-3K, and Vav/Rho-family G-protein, to mediate degranulation in effector cells.
Fig.2 NK cells mediate the three main signaling pathways involved in ADCC. ²
Activation of the relevant signaling pathways triggers an increase in intracellular calcium ions, the involvement of microtubule tissue centers (MTOCs), and the polarization and release of perforin/granzyme-containing organelles.
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Creative Biolabs provides services for targets including but not limited to: CD20, HER2, EGFR, CD19, BAFF, BCMA, PD-1/PD-L1, CD25, CD38, etc., and the experimental protocols cover conventional target cell lines, engineered cell lines with overexpressed targets, artificially constructed effector cell lines, etc. We provide customized protocols according to your experimental needs and deliver experimental results quickly. If you are interested, please contact us today!
Creative Biolabs provides luciferase-based ADCC assay. This Jurkat cell based assay is pioneered by Creative Biolabs, and the methodology is very well accepted by the field. See attached ADCC Reporter Assay Protocol for further details.
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