Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay
Summary
Here, we present a protocol for an antibody-dependent, cell-mediated cytotoxicity (ADCC) assay using an ADCC bioassay kit. This method offers a valuable tool for elucidating the ADCC mechanism and evaluating the therapeutic potential of antibodies in cancer immunotherapy.
Abstract
The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in cancer immunotherapy. Evaluating ADCC activity in cancer cells is essential for the development and optimization of antibody-based treatments. Here, we propose a methodological approach of utilizing an ADCC bioassay kit for quantitative assessment of ADCC reaction using thyroid cancer cells as effector cells. The protocol involves the co-culture of effector cells with target cancer cells in different ratios in the presence of a therapeutic antibody. The ADCC bioassay kit used in this experiment includes the genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. Upon the binding of the surface antigen on the target cells with the antibodies and effector cells, the effector cells release luciferase, enabling quantification of cytotoxicity through measurement of luminescence signal. In contrast to conventional ADCC assays, this method proved the binding of target antigen with antibodies and effector cells, which can produce reliable results in a short period.
Introduction
Antibody-dependent, cell-mediated cytotoxicity (ADCC) is an important mechanism by which antibodies exert immune-mediated cell-killing effects1,2,3. The immune cells are activated by binding to the therapeutic antibody, which interacts with surface antigens of the target cells to release granzymes, perforin, leading to the target cell death. These immune cells include natural killer (NK) cells and neutrophils2,3,4,5,6,7. The ADCC assay has become an important tool to evaluate the efficacy of therapeutic antibody8,9.
In the conventional ADCC assay, peripheral blood mononuclear cells (PBMCs) or natural killer cells are used as effector cells to monitor the efficacy of a therapeutic antibody by quantitating the target's cell death rate. Our method uses an ADCC bioassay kit that includes genetically engineered effector cells expressing a luciferase reporter gene under the control of Nuclear Factor of Activated T-cell (NFAT) response elements. We then quantify the binding of the surface antigen on the target cells with the antibody and the effector cells. This method is based on the ADCC reaction occurring in a short period without requiring human PBMC cells. The experimental steps include the co-culture of effector cells with target cells in the presence of therapeutic antibodies.
During incubation, the therapeutic antibody binds to the target antigen on the surface of the target cells, which leads to the binding of the effector cells and the Fc fragment of an antibody. This activates the NFAT response element and releases luminescence signals for the quantitative assessment of the ADCC reaction.
Before performing the experiment, the expression of the target antigen in the target cells must be confirmed by either flow cytometry or western blotting. Target cells are cultured and passaged into 96-well plates for 24 h before the experiment. Different concentrations of a therapeutic antibody are added together with different cell counts of effector cells to achieve the calculated effector-to-target cell ratio.
Key steps in this method include (1) preparation of target cells and effector cells, (2) effector-to-target cell ratios, (3) Preparation of different concentrations of the antibody, and (4) varying duration of incubation. After the incubation, luminescence signals are measured using a luminometer, providing a quantitative readout of ADCC activity. Compared to other methods for measuring ADCC, this method is relatively simple to operate, and the results are accurate.
The ADCC reporter bioassay indicates the binding of the target antigen, therapeutic antibody, and immune cells in the ADCC pathway activation. This binding activates gene transcription through the NFAT pathway in the effector cells-engineered Jurkat cells with stably expressing FcγRIIIa receptor, the V158 (high-affinity) variant. The NFAT response element mediates the expression of luciferase in the effector cells10,11. The biological activity of the antibody in the Mechanism of action (MOA) of ADCC is quantified through the luciferase signal produced from the NFAT pathway. Luciferase signal in the effector cells-FcγRIIIa receptor-expressing Jurket cells-is quantified using a luminescence reader (Figure 1). The signal-to-noise ratio of the assay is high.
Protocol
Representative Results
Discussion
Here, we have presented the ADCC Bioassay method for assessing the ADCC reaction of a therapeutic antibody. The method is straightforward and employs a simple “add-mix-read” format for measurement.
Before doing the experiment, the expression of the target antigen in the target cells must be confirmed by either flow cytometry or western blotting. Flow cytometry will be a better tool to detect the surface antigen. However, using flow cytometry can stress the cells, causing apoptosis and affecti…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful to Prof. Zeng (IMCB, A*STAR) for supporting this work. This study was supported by the Youth Foundation of National Natural Science Foundation of China (NSFC) (82202231), and the Medical and Health Science and Technology Project of Zhejiang Province, China (2021KY110,2024KY824).
Materials
| 0.5% Trypsin-EDTA | Gibco | 15400-054 | Dilute 10x in PBS to make 0.05% Trypsin |
| 1x Tris Buffer Saline (TBS) | 1st BASE | BUF-3030-1X1L | For membrane washing in western blotting |
| 1.5 M Tris Buffer, pH 8.8 | 1st BASE | BUF-1419-1L-pH8.8 | For SDS gel preparation |
| 2-Mercaptoethanol | Sigma Aldrich | M7522-100ML | For sample preparation of western blotting |
| 30% Acrylamide/Bis solution | Bio-Rad | #1610158 | For SDS gel preparation |
| 4x Laemmli Buffer | Bio-Rad | #1610747 | For sample preparation of western blotting |
| 96-well white polystyrene microplate with clear flat bottom | Corning Incorporated | 3610 | For ADCC assay |
| ADCC Bioassay Effector cells (0.65 mL) | Promega | G7011 | Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 1 vial |
| ADCC reporter bioassay core kit | Promega | G7010 | Mentioned as ADCC bioassay kit for ADCC assay in this experiment |
| Ammonium Persulfate | Sigma Aldrich | A3678-25G | For SDS gel preparation |
| Bevacizumab (Humanized Anti VEGF-antibody) | MVASI | – | Use as negative control antibody in ADCC asssay |
| BHT-101 | Leibniz Institute DSMZ | ACC279 | Human anaplastic papillary thyroid cancer cell line |
| Bio-Glo Luciferase Assay Buffer | Promega | G7941 | Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 10 mL |
| Bio-Glo Luciferase Assay Substrate (Lyophilized) | Promega | G7941 | Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 1 vial |
| Cell scraper | GenFollower | GD00235 | To remove cell from culture flask |
| Cetuximab (Chimeric anti-EGFR antibody) | ERBITUX | – | Use as therapeutic antibody in ADCC assay |
| Chemiluminescent HRP substrate | Merck Millipore | WBKLS0500 | For protein detection in western blotting |
| Distilled water | Gibco | 15230-162 | For SDS gel preparation |
| Fetal Bovine Serum (FBS) | Gibco | 10270-106 | Culture media supplement |
| iBright CL1500 imaging system | Thermo Scientific | 2462621100038 | For protein detection in western blotting |
| L-glutamine, 200 mM | Gibco | 25030-081 | Culture media supplement |
| Low IgG Serum | Promega | G7110 | Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 4 mL |
| Megafuge 8R | Thermo Scientific | 42876589 | Centrifuge |
| Mouse anti-EGFR monoclonal antibodies | BD Biosciences | 610016 | Primary antibody in western blotting |
| Mouse anti-VEGFR monoclonal antibodies | BD Biosciences | 571194 | Primary antibody in western blotting |
| non-enzymatic cell dissociation buffer | Sigma Aldrich | C5789-100ML | For cell harvesting from T75 flask |
| Penicillin-Streptomycin | PAN Biotech | P06-07100 | Antibacterial for culture media |
| Phosphate Buffered Saline (PBS), pH 7.2, Sterile filtered | 1st BASE | CUS-2048-1x1L | Use as washing solution for cells |
| Pierce BCA assay kit | Thermo Scientific | 23225 | To measure protein concentration |
| Protease and phosphatase inhibitor | Thermo Scientific | A32959 | For protein digestion in sample preparation for western blotting |
| PVDF membrane (Immobilin-P) | Merck Millipore | IPVH00010 | For protein transfer in western blotting |
| Rabbit anti-mouse IgG, Fcγ HRP-conjugated secondary antibody | Jackson ImmunoResearch | 315-035-046 | Secondary antibody in western blotting |
| Roswell Park Memorial Institute (RPMI) medium | Capricorn Scientific | RPMI-XA | Cell culture media |
| RPMI-1640 | Promega | G7080 | Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 36 mL |
| Skim milk powder | Merck Millipore | 70166-500G | For membrane blocking in western blotting |
| Sodium Dodecyl Sulfate | 1st BASE | BIO-2050-500g | For SDS gel preparation |
| SW-1736 | Cytion | 300453 | Human thyroid squamous cell cancer cell line |
| T75 culture flasks | SPL Lifesciences | 70075 | Cell culture flask |
| Tecan Multimode Reader model Spark 10M | Tecan | 1607000294 | for luminicence quantification |
| TEMED | Bio-Rad | #1610801 | For SDS gel preparation |
| Tween-20 | Promega | H5151 | For membrane washing in western blotting |
| Vi-cell XR cell viability analyzer | Beckman Coulter | AL15072 | Cell counter |
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