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,....

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 the.......

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.......

Materials

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