Name: Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay (Video) | JoVE Journal
Uploaded: 2024-09-13T14:20:00.000+00:00
Duration: 5 min 21 s
Description: Learn antibody-dependent cell-mediated cytotoxicity assessment for cancer immunotherapy using ADCC reporter bioassay. This rapid method enables accurate therapeutic antibody screening within one day.

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5 minutes

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).

1. Detection of EGFR and VEGFR expression in target cells
NOTE: Use western blotting to detect the expression of the target antigen in the target cells.
<ol>
	<li>Sample preparation
	<ol>
		<li>Culture the cells (BHT-101 and SW-1736 human thyroid cancer cell lines) in T75 flasks using Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% antibiotics (Penicillin-Streptomycin).</li>
		<li>Harvest the cells when they are 80% confluent by removing the culture media and washing the cells with PBS. Add 5 mL of PBS and scrap the cells using a cell scraper to remove the cells from the culture flask. Transfer the cells into a 15 mL conical tube.</li>
		<li>Count the cells with a cell counter.</li>
		<li>Aliquot 5 &#215; 106 of BHT-101 and SW-1736 cells into 15 mL conical tubes.</li>
		<li>Spin down the cells at 135 &#215; g for 5 min at room temperature. Remove the supernatant PBS.</li>
		<li>Lyse the cell pellet by adding 100 &#181;L of RIPA lysis buffer containing protease-phosphatase inhibitors for 5 min on ice. Transfer the lysate to a 2 mL tube, centrifuge at 15,000 &#215; g for 15 min, and transfer the supernatant into new 2 mL tube.</li>
		<li>Measure the protein concentration of the cell lysate by using a bicinchoninic acid (BCA) assay kit.</li>
		<li>Aliquot 70 &#181;g of protein from cell lysate from each sample, add 2x Laemmli sample buffer supplemented with 10% 2-mercaptoethanol, and boil the samples at 100 &#176;C for 5 min for denaturation of samples.</li>
	</ol>
	</li>
	<li>Gel electrophoresis and membrane transfer
	<ol>
		<li>Prepare 8% sodium dodecyl sulfate (SDS) polyacrylamide gel using distilled water (4.7 mL), 30% acrylamide (2.7 mL), 1.5 M Tris Buffer, pH 8.8 (2.5 mL), 10% sodium dodecyl sulfate (0.1 mL), 10% ammonium persulfate (100 &#181;L), and TEMED (10 &#181;L).</li>
		<li>Run 70 &#181;g of the denatured samples in the 8% SDS-polyacrylamide gel at 80 V for 20 min and at 120 V for 100 min.</li>
		<li>Transfer the protein on the gel to a polyvinylidene fluoride (PVDF) membrane at 100 V for 90 min.</li>
		<li>After transferring, block the membrane with 7% skim milk diluted in TBS-T buffer (20 mM Tris pH 7.6, 140 mM NaCl, 0.2% Tween-20) for 1 h.</li>
	</ol>
	</li>
	<li>Antibody incubation
	<ol>
		<li>Incubate the blocked membrane with mouse anti-Epidermal Growth Factor Receptor (EGFR) and rabbit anti-Vascular Endothelial Growth Factor Receptor (VEGFR) monoclonal antibodies (diluted at 1:1,000 in 7% skim milk) overnight at 4 &#176;C.</li>
		<li>After incubation, wash the membrane thoroughly with Tris-buffered saline-Tween (TBS-T) buffer (20 mM Tris pH 7.6, 140 mM NaCl, 0.2% Tween-20) for 7 min and repeat 4x.</li>
		<li>Incubate the membrane with the HRP-conjugated secondary antibody (diluted at 1:4,000 in 7% skim milk (TBS-T)) for 1 h.</li>
		<li>After incubation, wash the membrane thoroughly with TBS-T buffer for 7 min and repeat 4 times.</li>
	</ol>
	</li>
	<li>Protein detection
	<ol>
		<li>Incubate the membrane in chemiluminescent HRP substrate prepared by mixing 1 mL of HRP substrate peroxide solution with 1 mL of HRP substrate Luminol Reagent (both reagents are from the chemiluminescent substrate kit) for 2 min.</li>
		<li>Visualize the membrane using an imaging system.</li>
	</ol>
	</li>
</ol>
2. Preparation of target cells
<ol>
	<li>Culture the target T cells, BHT-101 and SW-1736 cells exponentially until 80% confluent in RPMI media supplemented with 10% FBS, 1% L-glutamine, and 1% antibiotics (Penicillin-Streptomycin) in T-75 flasks.</li>
	<li>After removing the media, wash the cells once with PBS. Incubate the cells with 1 ml of non-enzymatic cell dissociation buffer for 5 min to dislodge the adherent cells. Add PBS to stop the reaction of the non-enzymatic cell dissociation buffer. Spin down the cells at 135 &#215; g for 5 min and add 5 mL of PBS. 
	NOTE: Here, we use a non-enzymatic cell dissociation buffer to preserve the integrity of membrane surface protein.</li>
	<li>Count the cells and seed them at 15,000 cells/wells in 96-well, white polystyrene microplates with a clear, flat bottom.</li>
</ol>
3. Preparation of varying concentrations of the therapeutic antibody
<ol>
	<li>To follow this protocol, use Cetuximab (chimeric anti-EGFR antibody) to bind EGFR and use Bevacizumab (humanized anti VEGF-A antibody) as a negative control.</li>
	<li>Prepare ADCC Bioassay buffer by adding 1.4 mL of low IgG serum into 33.6 mL of RPMI-1640 (supplied in the kit).</li>
	<li>Use ADCC Bioassay buffer for the dilution of antibodies. Prepare 400 &#181;L of each antibody in three different concentrations: 30 &#181;g/mL, 3 &#181;g/mL, and 0.3 &#181;g/mL (3x concentration) to get the final concentration of 10 &#181;g/mL, 1 &#181;g/mL, and 0.1 &#181;g/mL for wide-spectrum coverage to detect the optimal dose. 
	NOTE: Final antibody concentrations are based on 25 &#181;L of cultured target cells, 25 &#181;L of effector cells, and 25 &#181;L of antibody in each well of a 96-well plate.</li>
</ol>
4. Preparation of effector cells
<ol>
	<li>Store the effector cells at -80 &#176;C before use.</li>
	<li>Preheat the ADCC Bioassay Buffer in a 37 &#176;C water bath for at least 30 min before use.</li>
	<li>Thaw the ADCC Bioassay Effector cells from -80 &#176;C cold storage by placing them in a 37 &#176;C water bath (approximately 2-3 min). Gently rock and visually inspect the vial, but do not invert it during the thawing process.</li>
	<li>Transfer 630ul of Effector cells to a 15 mL tube containing 3.6 mL of ADCC Assay Buffer. Mix well by gently inverting the tube 2x.</li>
</ol>
5. Incubation of effector cells with antibody and target cells
<ol>
	<li>After overnight incubation, remove the media from the target cells (15,000 cancer cells per well) and add 25 &#181;L of the ADCC Bioassay buffer and 25 &#181;L of Cetuximab (EGFR antagonist) and Bevacizumab (VEGF antagonist) to get the final concentration of 0.1 &#181;g/mL, 1 &#181;g/mL, or 10 &#181;g/mL of the target cells in each well according to Figure 2.</li>
	<li>Add 75,000 effector cells in 25 &#181;L of ADCC bioassay buffer per well into the 96-well microplate containing target cells. The wells labeled as No mAb act as no antibody control (Figure 2).</li>
	<li>Add ADCC Bioassay buffer to the wells labeled as AB (ADCC Bioassay buffer) for blank control.</li>
	<li>Incubate the plate for 6 h.</li>
</ol>
6. Quantitative readout of ADCC activity
<ol>
	<li>Prepare the luciferase assay reagent 4 h before the measurement by adding Luciferase Assay buffer to the Luciferase Assay Substrate (lyophilized).</li>
	<li>After 6 h of incubation of target cells, antibody, and effector cells, add 75 &#181;L of the luciferase assay reagent to each well and incubate for 30 min.</li>
	<li>Following the incubation period, measure the luminescence signal in each well by using a luminometer, providing a quantitative readout of ADCC activity.</li>
	<li>For data analysis, calculate the fold Induction as follows: 
	Fold induction = RLU (antibody induced - background) / RLU (no antibody control - background). 
	Where RLU is relative luminescence units; Antibody induced are wells B3 to B8 and C3 to C8; No antibody control = B2 and C2; Background = Average RLU from well A2 to A5 (Figure 2).</li>
</ol>

Thyroid Cancer Cells as Effectors in an ADCC Bioassay for Cancer Immunotherapy

<ol><li>Zahavi, D., AlDeghaither, D., O'Connell, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancing+antibody-dependent+cell-mediated+cytotoxicity%3A+a+strategy+for+improving+antibody-based+immunotherapy%5BTitle%5D)+AND+%22Antib+Ther%22%5BJournal%5D)">Enhancing antibody-dependent cell-mediated cytotoxicity: a strategy for improving antibody-based immunotherapy.</a> Antib Ther. 1 (1), 7-12 (2018).</li>
<li>Fenis, A., Demaria, O., Gauthier, L., Vivier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((New+immune+cell+engagers+for+cancer+immunotherapy%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">New immune cell engagers for cancer immunotherapy.</a> Nat Rev Immunol. 24 (7), 471-486 (2024).</li>
<li>Pinto, S., Pahl, J., Schottelius, A., Carter, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reimagining+antibody-dependent+cellular+cytotoxicity+in+cancer%3A+the+potential+of+natural+killer+cell+engagers%5BTitle%5D)+AND+%22Trends+Immunol%22%5BJournal%5D)">Reimagining antibody-dependent cellular cytotoxicity in cancer: the potential of natural killer cell engagers.</a> Trends Immunol. 43 (11), 932-946 (2022).</li>
<li>Ochoa, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody-dependent+cell+cytotoxicity%3A+immunotherapy+strategies+enhancing+effector+NK+cells%5BTitle%5D)+AND+%22Immunol+Cell+Biol%22%5BJournal%5D)">Antibody-dependent cell cytotoxicity: immunotherapy strategies enhancing effector NK cells.</a> Immunol Cell Biol. 95 (4), 347-355 (2017).</li>
<li>Wang, W., Erbe, A. K., Hank, J. A., Morris, Z. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NK+cell-mediated+antibody-dependent+cellular+cytotoxicity+in+cancer+immunotherapy%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">NK cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy.</a> Front Immunol. 6, 368(2015).</li>
<li>Chung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+in+vitro+antibody-dependent+cell-mediated+cytotoxicity+activity+of+therapeutic+antibodies+-+impact+of+effector+cells%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Characterization of in vitro antibody-dependent cell-mediated cytotoxicity activity of therapeutic antibodies - impact of effector cells.</a> J Immunol Methods. 407, 63-75 (2014).</li>
<li>Shimasaki, N., Jain, A., Campana, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NK+cells+for+cancer+immunotherapy%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">NK cells for cancer immunotherapy.</a> Nat Rev Drug Discov. 19 (3), 200-218 (2020).</li>
<li>Cheng, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+robust+reporter-based+ADCC+assay+with+frozen%2C+thaw-and-use+cells+to+measure+Fc+effector+function+of+therapeutic+antibodies%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Development of a robust reporter-based ADCC assay with frozen, thaw-and-use cells to measure Fc effector function of therapeutic antibodies.</a> J Immunol Methods. 414, 69-81 (2014).</li>
<li>Parekh, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+and+validation+of+an+antibody-dependent+cell-mediated+cytotoxicity-reporter+gene+assay%5BTitle%5D)+AND+%22MAbs%22%5BJournal%5D)">Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay.</a> MAbs. 4 (3), 310-318 (2012).</li>
<li>Hogarth, P. M., Pietersz, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fc+receptor-targeted+therapies+for+the+treatment+of+inflammation%2C+cancer+and+beyond%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond.</a> Nat Rev Drug Discov. 11 (4), 311-331 (2012).</li>
<li>Chung, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+evaluation+of+fucose+reducing+effects+in+a+humanized+antibody+on+Fcgamma+receptor+binding+and+antibody-dependent+cell-mediated+cytotoxicity+activities%5BTitle%5D)+AND+%22MAbs%22%5BJournal%5D)">Quantitative evaluation of fucose reducing effects in a humanized antibody on Fcgamma receptor binding and antibody-dependent cell-mediated cytotoxicity activities.</a> MAbs. 4 (3), 326-340 (2012).</li>
<li>Promega. ADCC Reporter Bioassay Core Kit Technical Manual. , 
 <a target="_blank" href="https://www.promega.sg/-/media/files/resources/protocols/technical-manuals/101/adcc-reporter-bioassay-core-kit-protocol.pdf?rev=bec36264c0b6470591ded081377d207d&sc_lang=en">https://www.promega.sg/-/media/files/resources/protocols/technical-manuals/101/adcc-reporter-bioassay-core-kit-protocol.pdf?rev=bec36264c0b6470591ded081377d207d&sc_lang=en</a> (2023).</li>
<li>Miller, A. S., Tejada, M. L., Gazzano-Santoro, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+measuring+antibody-dependent+cell-mediated+cytotoxicity+in+vitro%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Methods for measuring antibody-dependent cell-mediated cytotoxicity in vitro.</a> Methods Mol Biol. 1134, 59-65 (2014).</li>
<li>Lo Nigro, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NK-mediated+antibody-dependent+cell-mediated+cytotoxicity+in+solid+tumors%3A+biological+evidence+and+clinical+perspectives%5BTitle%5D)+AND+%22Ann+Transl+Med%22%5BJournal%5D)">NK-mediated antibody-dependent cell-mediated cytotoxicity in solid tumors: biological evidence and clinical perspectives.</a> Ann Transl Med. 7 (5), 105(2019).</li>
<li>Gómez Román, V. R., Murray, J. C., Weiner, L. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aG%C3%B3mez+Rom%C3%A1n%2C+VR+%22Antibody+Fc%22">Antibody Fc</a>. Ackerman, M. E., Nimmerjahn, F. , Academic Press. 1-27 (2014).</li>
</ol>

All authors declare no conflicts of interest.

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 affecting the viability of the cell and, therefore, the overall analysis. In addition, it costs more than western blotting. In this experiment, we used western blotting as a faster and more cost-effective method because targeted antigens are known cell surface antigens. 
Engineered Jurkat cells, which stably expressed the Fc&#947;RIIIa receptor, the V158 (high affinity) variant, and an NFAT response element that drives the expression of firefly luciferase, were used as effector cells. Activation of the NFAT pathway results in the production of luciferase, and its activity is quantified in the form of luminescence readout. The signal also represents the activity of ADCC.
First, target cells (T) are incubated with effector cells (E) in the presence of varying amounts of antibody in 96-well plates for 6 h at 37 &#176;C in a humidified CO2 incubator. The effector:target ratio of 5:1 is used to optimize the signal. For example, 75,000 effector cells were added to 15,000 target cells. At the end of incubation, Bio-Glo luciferase assay reagent is added to each well, incubated for 30 min, and the luminescence (RLU, relative luciferase units) is measured using a luminescence plate reader. 
The key critical steps in this protocol are: (1) The handling of effector cells. The cell vial should not be inverted during the thawing process, rock gently, and must be used immediately after thawing to prevent unwanted cell death or affecting the performance of biological detection. (2). It is important to use white polystyrene 96-well microplates with clear, flat bottom for luminescence measurements because the luminescence is captured from the bottom of the reader. 
In this study, we observed the stronger and weaker ADCC reactions of the anti-EGFR antibody, cetuximab, depending on the amount of target cells and antibody concentration. Some reasons for handling the calibration of weak ADCC reactions: (1) The readings of the ADCC reporter bioassay come from effector cells (E), with a constant number of 75,000 cells per well in the ADCC reaction system of this study. Therefore, optimizing the quality of target cells (T) is one aspect that can be improved. The E:T ratio in this study was 5:1, which could be adjusted up to 20:1. (2) Antibody concentration is also one of the important factors affecting ADCC reaction. Adjustments can be made through serial dilution of antibodies to explore the optimal concentration range, thus achieving maximum response in the ADCC report. (3) The incubation time of antibodies, target cells, and effector cells is also crucial for experimental results. In this study, we incubated for 6 h, which can be extended up to 24 h to achieve the optimal ADCC reaction. (4) Additionally, the concentration of the buffer solution (low IgG) for ADCC assay also needs to be explored. The optimal serum concentration for ADCC response can be achieved within the range of 1% to 10%.
This experimental method is rapid and straightforward, allowing completion within one day through a simple "add-mix-read" protocol. The results are stable and amenable to batch testing. However, this bioassay kit does not detect cell death like conventional ADCC methods that use PBMC or NK cells as effector cells12,13. Instead, it uses Fc&#947;RIIIa receptor expressed Jurkat cells as effector cells, which will cost more because the cells are genetically modified. In comparison to traditional ADCC experiments13,14, it eliminates the requirement of blood donations from healthy individuals and avoids the complex process of extracting immune cells, thus mitigating individual variations that could impact the results. The detected luminescence signals are from the binding of effector cells to target cells, not from the actual death of target cells. Therefore, there may be discrepancies compared to standard ADCC detection methods. Furthermore, cell dissociation buffer, which is more expensive than trypsin, is used to detach the adherent cells to maintain the membrane integrity.
ADCC reporter gene analysis demonstrates excellent accuracy and stability, serving as an efficacy analysis method for the mass detection of therapeutic antibody drugs. It can also function as a critical analysis method for the characterization of therapeutic molecules and process development15.
In summary, ADCC is an important immune mechanism, and the quantitative detection of ADCC holds great importance in the field of immunotherapy. This experimental method offers an effective means for the quantitative measurement of ADCC15.

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&#39;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 Fc&#947;RIIIa 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-Fc&#947;RIIIa receptor-expressing Jurket cells-is quantified using a luminescence reader (Figure 1). The signal-to-noise ratio of the assay is high.

<table><tbody><tr><td>0.5% Trypsin-EDTA</td><td>Gibco</td><td>15400-054</td><td>Dilute 10x in PBS to make 0.05% Trypsin</td></tr><tr><td>1x Tris Buffer Saline (TBS)</td><td>1st BASE</td><td>BUF-3030-1X1L</td><td>For membrane washing in western blotting</td></tr><tr><td>1.5 M Tris Buffer, pH 8.8</td><td>1st BASE</td><td>BUF-1419-1L-pH8.8</td><td>For SDS gel preparation</td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma Aldrich</td><td>M7522-100ML</td><td>For sample preparation of western blotting</td></tr><tr><td>30% Acrylamide/Bis solution</td><td>Bio-Rad</td><td>#1610158</td><td>For SDS gel preparation</td></tr><tr><td>4x Laemmli Buffer</td><td>Bio-Rad</td><td>#1610747</td><td>For sample preparation of western blotting</td></tr><tr><td>96-well white polystyrene microplate with clear flat bottom</td><td>Corning Incorporated</td><td>3610</td><td>For ADCC assay</td></tr><tr><td>ADCC Bioassay Effector cells (0.65 mL)</td><td>Promega</td><td>G7011</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 1 vial</td></tr><tr><td>ADCC reporter bioassay core kit</td><td>Promega</td><td>G7010</td><td>Mentioned as ADCC bioassay kit for ADCC assay in this experiment</td></tr><tr><td>Ammonium Persulfate</td><td>Sigma Aldrich</td><td>A3678-25G</td><td>For SDS gel preparation</td></tr><tr><td>Bevacizumab (Humanized Anti VEGF-antibody)</td><td>MVASI</td><td>-</td><td>Use as negative control antibody in ADCC asssay</td></tr><tr><td>BHT-101</td><td>Leibniz Institute DSMZ</td><td>ACC279</td><td>Human anaplastic papillary thyroid cancer cell line&amp;nbsp;</td></tr><tr><td>Bio-Glo Luciferase Assay Buffer</td><td>Promega</td><td>G7941</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 10 mL</td></tr><tr><td>Bio-Glo Luciferase Assay Substrate (Lyophilized)</td><td>Promega</td><td>G7941</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 1 vial</td></tr><tr><td>Cell scraper</td><td>GenFollower</td><td>GD00235</td><td>To remove cell from culture flask</td></tr><tr><td>Cetuximab (Chimeric anti-EGFR antibody)</td><td>ERBITUX</td><td>-</td><td>Use as therapeutic antibody in ADCC assay</td></tr><tr><td>Chemiluminescent HRP substrate</td><td>Merck Millipore</td><td>WBKLS0500</td><td>For protein detection in western blotting</td></tr><tr><td>Distilled water</td><td>Gibco</td><td>15230-162</td><td>For SDS gel preparation</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270-106</td><td>Culture media supplement</td></tr><tr><td>iBright CL1500 imaging system</td><td>Thermo Scientific</td><td>2462621100038</td><td>For protein detection in western blotting</td></tr><tr><td>L-glutamine, 200 mM</td><td>Gibco</td><td>25030-081</td><td>Culture media supplement</td></tr><tr><td>Low IgG Serum</td><td>Promega</td><td>G7110</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 4 mL</td></tr><tr><td>Megafuge 8R</td><td>Thermo Scientific</td><td>42876589</td><td>Centrifuge</td></tr><tr><td>Mouse anti-EGFR monoclonal antibodies</td><td>BD Biosciences</td><td>610016</td><td>Primary antibody in western blotting</td></tr><tr><td>Mouse anti-VEGFR monoclonal antibodies</td><td>BD Biosciences</td><td>571194</td><td>Primary antibody in western blotting</td></tr><tr><td>non-enzymatic cell dissociation buffer</td><td>Sigma Aldrich</td><td>C5789-100ML</td><td>For cell harvesting from T75 flask</td></tr><tr><td>Penicillin-Streptomycin</td><td>PAN Biotech</td><td>P06-07100</td><td>Antibacterial for culture media</td></tr><tr><td>Phosphate Buffered Saline (PBS), pH 7.2, Sterile filtered</td><td>1st BASE</td><td>CUS-2048-1x1L</td><td>Use as washing solution for cells</td></tr><tr><td>Pierce BCA assay kit</td><td>Thermo Scientific</td><td>23225</td><td>To measure protein concentration</td></tr><tr><td>Protease and phosphatase inhibitor</td><td>Thermo Scientific</td><td>A32959</td><td>For protein digestion in sample preparation for western blotting</td></tr><tr><td>PVDF membrane (Immobilin-P)</td><td>Merck Millipore</td><td>IPVH00010</td><td>For protein transfer in western blotting</td></tr><tr><td>Rabbit anti-mouse IgG, Fc&amp;gamma; HRP-conjugated secondary antibody</td><td>Jackson ImmunoResearch</td><td>315-035-046</td><td>Secondary antibody in western blotting</td></tr><tr><td>Roswell Park Memorial Institute (RPMI) medium</td><td>Capricorn Scientific</td><td>RPMI-XA</td><td>Cell culture media</td></tr><tr><td>RPMI-1640</td><td>Promega</td><td>G7080</td><td>Includes in ADCC reporter bioassay core kit (Promega G7010), 1 x 36 mL</td></tr><tr><td>Skim milk powder</td><td>Merck Millipore</td><td>70166-500G</td><td>For membrane blocking in western blotting</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>1st BASE</td><td>BIO-2050-500g</td><td>For SDS gel preparation</td></tr><tr><td>SW-1736</td><td>Cytion</td><td>300453</td><td>Human thyroid squamous cell cancer cell line</td></tr><tr><td>T75 culture flasks</td><td>SPL Lifesciences</td><td>70075</td><td>Cell culture flask</td></tr><tr><td>Tecan Multimode Reader model Spark 10M</td><td>Tecan</td><td>1607000294</td><td>for luminicence quantification</td></tr><tr><td>TEMED</td><td>Bio-Rad</td><td>#1610801</td><td>For SDS gel preparation</td></tr><tr><td>Tween-20</td><td>Promega</td><td>H5151</td><td>For membrane washing in western blotting</td></tr><tr><td>Vi-cell XR cell viability analyzer</td><td>Beckman Coulter</td><td>AL15072</td><td>Cell counter</td></tr></tbody></table>

assessing antibody-dependent, cell-mediated cytotoxicity in cancer cells using antibody-dependent cell-mediated cytotoxicity reporter bioassay

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

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.

The expression of EGFR and VEGFR in the target BHT-101 and SW-1736 cells was detected using western blotting. EGFR expression was detected in both BHT-101 and SW-1736 cells but not VEGFR expression (Figure 3).
Using the ADCC bioassay kit, we detected the ADCC reaction of the anti-EGFR antibody, cetuximab, using EGFR-positive cell lines, BHT-101 and SW-1736, as target cells. Bevacizumab, a VEGF inactivator, was used as a negative control antibody. Different concentrations of antibodies and effector cells were incubated with the target cells. The luminescence signal was detected using a plate reader (Figure 2). A higher fold of ADCC activity induction was found in Cetuximab (anti-EGFR antibody) groups, but no ADCC activity was seen in Bevacizumab (VEGF antagonist) for both cell lines. This demonstrated the ADCC effect targeting EGFR extracellular membrane antigens for both cell lines. By using this method, the ADCC reaction was validated in the presence of Cetuximab in two anaplastic thyroid cancer cell lines (BHT101 and SW1736) (Figure 4).
<img alt="Antibody-dependent cell cytotoxicity (ADCC) diagram with antigen-antibody interaction and luminescence." class="xfigimg" src="/files/ftp_upload/67077/67077fig01.jpg" data-altupdatedat="1788304762000" data-alt="Figure 1"> 
Figure 1: A schematic diagram showing the ADCC reaction with the ADCC bioassay kit. Antigen-binding sites in the therapeutic antibody bind to surface antigens in target cells. This binding leads to the binding of the Fc portion of the antibody to the FcƳRIIIa receptors of effector cells, which have been genetically engineered with NFAT-RE luc, producing a luminescence signal in the effector cells. This figure was modified from the ADCC Reporter Bioassay Core kit manual with permission12. Abbreviations: ADCC = Antibody-dependent cell-mediated cytotoxicity; Fc = Fragment crystallizable region; NFAT = Nuclear Factor of Activated T-cell; RE = Response element; luc = Luciferase. <a href="https://www.jove.com/files/ftp_upload/67077/67077fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="ADCC bioassay results table, VEGFR, EGFR analysis, BHT101, SW1736 data, antibody effects shown." class="xfigimg" src="/files/ftp_upload/67077/67077fig02.jpg" data-altupdatedat="1788304762000" data-alt="Figure 2"> 
Figure 2: Luminescence reading and experimental diagram of 96-well plate design of the ADCC co-culture system. Luminescence signals are read by a luminescence reader. Row A: 1st-9th wells contain ADCC Bioassay Buffer. Row B: 3rd-8th wells contain BHT101's ADCC reaction system (target cells+ antibody + effector cells). The 2nd well is without antibody. The 9th well contains just ADCC Bioassay buffer. Row C: 3rd-8th wells contain SW-1736's ADCC reaction system (target cell+ antibody + effector cells). The 2nd well is without antibody. The 9th well contains just ADCC Bioassay buffer. Row D: 1st-9th wells contain Assay Buffer. Abbreviations: ADCC = Antibody-dependent cell-mediated cytotoxicity; AB = ADCC Bioassay buffer; VEGFR = Vascular Epithelial Growth Factor Receptor; EGFR = Epidermal Growth Factor Receptor; mAb = monoclonal antibody. <a href="https://www.jove.com/files/ftp_upload/67077/67077fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Western blot shows EGFR, VEGFR, B-actin protein levels in cell lines BHT-101, SW-1736." class="xfigimg" src="/files/ftp_upload/67077/67077fig03.jpg" data-altupdatedat="1788304762000" data-alt="Figure 3"> 
Figure 3: Detection of EGFR and VEGFR expression in BHT-101 and SW-1736 thyroid carcinoma using western blotting. Expression of EGFR, but not VEGFR, was detected in both cell lines. B-actin was used as the loading control. Abbreviations: EGFR = epidermal growth factor receptor; VEGFR = vascular epidermal growth factor. <a href="https://www.jove.com/files/ftp_upload/67077/67077fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Luminescence intensity graph; Cetuximab and Bevacizumab effects; BHT 101, SW 1736 cell analysis." class="xfigimg" src="/files/ftp_upload/67077/67077fig04.jpg" data-altupdatedat="1788304762000" data-alt="Figure 4"> 
Figure 4: Fold induction of ADCC activity. (A) Thyroid cancer cell line BHT-101 exhibits an ADCC effect in the presence of the anti-EGFR antibody Cetuximab. (B) The thyroid cancer cell line SW-1736 exhibits an ADCC effect in the presence of the anti-EGFR antibody Cetuximab. <a href="https://www.jove.com/files/ftp_upload/67077/67077fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

The method for antibody-dependent, cell-mediated cytotoxicity (ADCC) represents an important tool to assess the efficacy of therapeutic antibodies in ...

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JoVE Journal

Immunology and Infection

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Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

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Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

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Measuring the Activation of Fc-Mediated Effector Functions by HA Antibodies

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Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

Real-time Cytotoxicity Assays in Human Whole Blood

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A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

This protocol presents an automated, image-based high-throughput technique to identify compounds modulating natural killer cell-mediated breast cancer cell killing in the presence of a therapeutic anti-HER-2 antibody.

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds

The use of cytokine release assay reference reagents allows for more reproducible and standardized in vitro safety profiles of immunotherapeutic monoclonal antibodies. Here we describe how cytokine release assays can be used alongside a reference reagent panel to predict the safety of some therapeutic monoclonal antibodies.

Using Reference Reagents to Confirm Robustness of Cytokine Release Assays for the Prediction of Monoclonal Antibody Safety

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

Learn antibody-dependent cell-mediated cytotoxicity assessment for cancer immunotherapy using ADCC reporter bioassay. This rapid method enables accurate therapeutic antibody screening within one day.

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay (Video) | JoVE Journal

Assessing Antibody-dependent, Cell-mediated Cytotoxicity in Cancer Cells using Antibody-Dependent Cell-Mediated Cytotoxicity Reporter Bioassay

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