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 effects^1,2,3. 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 neutrophils^2,3,4,5,6,7. The ADCC assay has become an important tool to evaluate the efficacy of therapeutic antibody^8,9.

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 cells^10,11. 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

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.

1. Sample preparation
   1. 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).
   2. 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.
   3. Count the cells with a cell counter.
   4. Aliquot 5 × 10⁶ of BHT-101 and SW-1736 cells into 15 mL conical tubes.
   5. Spin down the cells at 135 × g for 5 min at room temperature. Remove the supernatant PBS.
   6. Lyse the cell pellet by adding 100 µL 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 × g for 15 min, and transfer the supernatant into new 2 mL tube.
   7. Measure the protein concentration of the cell lysate by using a bicinchoninic acid (BCA) assay kit.
   8. Aliquot 70 µg of protein from cell lysate from each sample, add 2x Laemmli sample buffer supplemented with 10% 2-mercaptoethanol, and boil the samples at 100 °C for 5 min for denaturation of samples.
2. Gel electrophoresis and membrane transfer
   1. 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 µL), and TEMED (10 µL).
   2. Run 70 µg of the denatured samples in the 8% SDS-polyacrylamide gel at 80 V for 20 min and at 120 V for 100 min.
   3. Transfer the protein on the gel to a polyvinylidene fluoride (PVDF) membrane at 100 V for 90 min.
   4. 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.
3. Antibody incubation
   1. 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 °C.
   2. 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.
   3. Incubate the membrane with the HRP-conjugated secondary antibody (diluted at 1:4,000 in 7% skim milk (TBS-T)) for 1 h.
   4. After incubation, wash the membrane thoroughly with TBS-T buffer for 7 min and repeat 4 times.
4. Protein detection
   1. 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.
   2. Visualize the membrane using an imaging system.

2. Preparation of target cells

1. 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.
2. 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 × 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.
3. Count the cells and seed them at 15,000 cells/wells in 96-well, white polystyrene microplates with a clear, flat bottom.

3. Preparation of varying concentrations of the therapeutic antibody

1. 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.
2. Prepare ADCC Bioassay buffer by adding 1.4 mL of low IgG serum into 33.6 mL of RPMI-1640 (supplied in the kit).
3. Use ADCC Bioassay buffer for the dilution of antibodies. Prepare 400 µL of each antibody in three different concentrations: 30 µg/mL, 3 µg/mL, and 0.3 µg/mL (3x concentration) to get the final concentration of 10 µg/mL, 1 µg/mL, and 0.1 µg/mL for wide-spectrum coverage to detect the optimal dose.
   NOTE: Final antibody concentrations are based on 25 µL of cultured target cells, 25 µL of effector cells, and 25 µL of antibody in each well of a 96-well plate.

4. Preparation of effector cells

1. Store the effector cells at -80 °C before use.
2. Preheat the ADCC Bioassay Buffer in a 37 °C water bath for at least 30 min before use.
3. Thaw the ADCC Bioassay Effector cells from -80 °C cold storage by placing them in a 37 °C water bath (approximately 2-3 min). Gently rock and visually inspect the vial, but do not invert it during the thawing process.
4. 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.

5. Incubation of effector cells with antibody and target cells

1. After overnight incubation, remove the media from the target cells (15,000 cancer cells per well) and add 25 µL of the ADCC Bioassay buffer and 25 µL of Cetuximab (EGFR antagonist) and Bevacizumab (VEGF antagonist) to get the final concentration of 0.1 µg/mL, 1 µg/mL, or 10 µg/mL of the target cells in each well according to Figure 2.
2. Add 75,000 effector cells in 25 µL 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).
3. Add ADCC Bioassay buffer to the wells labeled as AB (ADCC Bioassay buffer) for blank control.
4. Incubate the plate for 6 h.

6. Quantitative readout of ADCC activity

1. Prepare the luciferase assay reagent 4 h before the measurement by adding Luciferase Assay buffer to the Luciferase Assay Substrate (lyophilized).
2. After 6 h of incubation of target cells, antibody, and effector cells, add 75 µL of the luciferase assay reagent to each well and incubate for 30 min.
3. Following the incubation period, measure the luminescence signal in each well by using a luminometer, providing a quantitative readout of ADCC activity.
4. 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).

Results

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).

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. Please click here to view a larger version of this figure.

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: 1^st-9^th wells contain ADCC Bioassay Buffer. Row B: 3^rd-8^th wells contain BHT101's ADCC reaction system (target cells+ antibody + effector cells). The 2^nd well is without antibody. The 9^th well contains just ADCC Bioassay buffer. Row C: 3^rd-8^th wells contain SW-1736's ADCC reaction system (target cell+ antibody + effector cells). The 2^nd well is without antibody. The 9^th well contains just ADCC Bioassay buffer. Row D: 1^st-9^th 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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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 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 FcγRIIIa 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 °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 cells^12,13. Instead, it uses FcγRIIIa receptor expressed Jurkat cells as effector cells, which will cost more because the cells are genetically modified. In comparison to traditional ADCC experiments^13,14, 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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