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. 2021 Jun 17;9(6):660.
doi: 10.3390/vaccines9060660.

Development of a Macrophage-Based ADCC Assay

Melissa B Uccellini  1   2 , Sadaf Aslam  1   2 , Sean T H Liu  1 , Fahmida Alam  1   2 , Adolfo García-Sastre  1   2   3   4
Affiliations

Development of a Macrophage-Based ADCC Assay

Melissa B Uccellini et al. Vaccines (Basel). .

Abstract

Fc-dependent effector functions are an important determinant of the in vivo potency of therapeutic antibodies. Effector function is determined by the combination of FcRs bound by the antibody and the cell expressing the relevant FcRs, leading to antibody-dependent cellular cytotoxicity (ADCC). A number of ADCC assays have been developed; however, they suffer from limitations in terms of throughput, reproducibility, and in vivo relevance. Existing assays measure NK cell-mediated ADCC activity; however, studies suggest that macrophages mediate the effector function of many antibodies in vivo. Here, we report the development of a macrophage-based ADCC assay that relies on luciferase expression in target cells as a measure of live cell number. In the presence of primary mouse macrophages and specific antibodies, loss of luciferase signal serves as a surrogate for ADCC-dependent killing. We show that the assay functions for a variety of mouse and human isotypes with a model antigen/antibody complex in agreement with the known effector function of the isotypes. We also use this assay to measure the activity of a number of influenza-specific antibodies and show that the assay correlates well with the known in vivo effector functions of these antibodies.

Keywords: ADCC; hemagglutinin; influenza; macrophage.

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Conflict of interest statement

The authors report no potential conflict of interest.

Figures

Figure 1
Figure 1
Macrophage-based ADCC assay. (A) Increasing numbers of MDCK or Raji target cells were incubated with D-luciferin substrate and luminescence was measured at 1 h post-addition. (B) MDCK-cH6/1 and Raji-cH6/1 cells were stained with HA-specific antibody 8H9 followed by anti-mouse Alexa 555. (C) MDCK target cells were incubated with 6F12 or isotype control Ab in the absence or presence of various ratios of macrophages and luminescence was measured at the indicated time points. No killing was observed in the absence of antigen expression. (D) MDCK-cH6/1 target cells were incubated as in C. Killing was observed in the presence of antigen expression and the specific antibody. (E) Raji target cells were incubated with the indicated antibodies. Killing was again observed with the specific antibody.
Figure 2
Figure 2
ADCC activity of a panel of HA-specific antibodies. (A) MCDK-cH6/1 target cells were incubated with the indicated antibodies at 1 ug/mL in the presence or absence of macrophages at a 1:3 ratio. ADCC activity was measured at 24 h. A number of antibodies showed ADCC activity on MDCK-cH6/1 target cells. (B) Raji-cH6/1 target cells were incubated as in A. Killing was only observed with CR9114 and the control antibody Rituximab. *** p < 0.001, **** p < 0.0001.
Figure 3
Figure 3
Binding of a panel of HA-specific antibodies. MDCK-cH6/1 and Raji-cH6/1 cells were incubated with the indicated antibodies followed by either human (top) or mouse (bottom) secondary antibody. GFP expression and cell surface binding were measured by flow cytometry.
Figure 4
Figure 4
ADCC activity of mouse and human Rituximab isotypes. Raji-cH6/1 target cells were incubated with the indicated antibodies at 1 ug/mL in the presence or absence of macrophages at a 1:3 ratio. ADCC activity was measured at 24 h. ** p < 0.01, **** p < 0.0001.
See this image and copyright information in PMC

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