APPLICATION NOTE

Quantification of anti-HER2 ADCC activity
using iLite® ADCC Assay Ready Cells
For research and professional use only. Not for use in diagnostic procedures.
This application note contains a suggested protocol and performance data.
Each individual laboratory must set up their own method and perform relevant validations.

Background
Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism whereby pathogenic cells arelysed by lymphocytes, most often Natural Killer (NK) cells. The mechanism involves binding ofantibodies to surface antigens on the pathogen. Crosslinking of these antibodies to NK cells throughthe binding of the Fc-portion to Fc receptors on the NK cells leads to activation of the NK cell andformation of an immune synapse with the pathogenic cell. The NK cell releases cytotoxic granules
containing granzymes and perforin into the synapse, leading to apoptosis of the targeted cell (1).
Breast cancer is the most common cancer in women worldwide, and the second most common cancer
overall. Survival rates have improved in the recent years, and stratification of patients into subgroups
has vastly improved treatment options for many patients. As an example, patients with HER2 positive
breast cancer generally have a poor prognosis, but treatment with trastuzumab, a monoclonal
antibody targeting the HER2 receptor, has shown to increase both overall survival and disease-freesurvival when given together with chemotherapy (2). Trastuzumab’s mechanism of action is mediatedin part by inducing ADCC when crosslinking HER2 positive cells with the patient's immune cells. The
drug was FDA approved for treatment of breast cancer patients in 1998, and many biosimilars arecurrently in development (3, 4).

Principle of the assay

The iLite® ADCC Assay Ready Cells are engineered cells that enable antibody-dependent cell-mediatedcytotoxicity (ADCC) to be examined through the specific expression of Firefly luciferase. When theantibodies of interest bind to the antigens on the surface of the target cell, the target-bound antibodies
will be presented to the Fc receptors (FcγRIIIa) on the effector cell. When the Fc-portion of the targetbound antibodies binds to the receptor, multiple cross-linking of the two cell types occurs. This will initiate
a signaling cascade which triggers the expression of Firefly luciferase (FL) in the effector cell. In thisapplication note, we describe the use of an effector cell line (iLite® ADCC Effector (V) Assay ReadyCells) that over-express FcγRIIIa and contain the FL reporter gene that responds to the principaltranscription factors that mediate signaling from the FcγRIIIa receptor, together with a positive target
cell line which over-expresses the surface antigen HER2 (iLite® HER2 (+) Target Assay Ready Cells).
iLite® ADCC Effector (V) Assay Ready Cells also contain the Renilla Luciferase (RL) reporter gene,
under the control of a constitutive promoter, that allows drug-induced FL activity to be normalized withrespect to the constitutive expression of RL. This renders assay results independent of variations in cellnumber, serum matrix effects, or lysis of the effector cells by the target cells. In addition, we also describe
the use of a negative control in the form of a target cell line depleted of HER2 expression (iLite® HER2(-) Target Assay Ready Cells). The Firefly luciferase signal can be measured in a luminometer following
addition and incubation of luciferase substrate. The Firefly luciferase signal is proportional to thefunctional activity of trastuzumab in the sample (Fig 1).

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

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APPLICATION NOTE
Material and equipment needed
Material and equipment

Suggested supplier

ReferenceiLite® ADCC Effector (V) Assay Ready CellsiLite® HER2 (+) Target Assay Ready Cells
iLite® HER2 (-) Target Assay Ready Cells
Diluent (RPMI 1640 + 9% heat inactivated FBS +1% Penicillin Streptomycin)

Svar Life Science
Svar Life Science
Svar Life Science
Gibco

Trastuzumab or analogues
Firefly/Renilla luciferase substrate

Roche
Promega

Plate; White walled micro well plate suitable forluminescence
Microplate Luminometer with appropriate reading
software – no filter on luminometer

PerkinElmer

BM5001
BM5011
BM501661870 (RPMI)
26140-079 (FBS)
15140-122(PenicillinStreptomycin)
NA
E2940, Dual-Glo®
Luciferase Assay
System6005680

Incubator, 37C with 5% CO2
Water bath, 37C
Single-channel and multi-channel pipettes with
polypropylene disposable tips
Polypropylene tubes or plate for dilution
Single-use polypropylene reservoir
Plate shaker
Timer

Contact Svar Life
Science for list ofrecommended
suppliers
NA
NA
NA

NA

NA
NA
NA
NA

NA
NA
NA
NA

NA
NA
NA

Preparation of calibrators (trastuzumab)

100
1000
1000
01

100.
1

0.
01

0.
00
01
0.
00
1

The ADCC effect of the trastuzumab antibody from Roche has successfully been measured incombination with a mix of ADCC Effector (V) Assay Ready Cells and HER2 (+) Target Assay ReadyCells. As a negative control, a combination of ADCC Effector (V) Assay Ready Cell and HER2 (-) Target
Assay Ready Cells was used. In the present assay an Effector:Target ratio of 4:1 has been used. The
optimal ratio is dependent on the antibody and target cells used and should be determined each time anew assay is set up. The table below shows recommended dilutions of trastuzumab when making an 8point calibration curve.
Trastuzumab
Calibrator Calibrator solution conc.
6000000(ng/mL)
5000000
14 0004000000
2267
3000000
327
2000000
42.7
Effector (V) and Target HER2 (+)
50.27
1000000
Effector (V) and Target HER2 (-)
60.027070.00089
80
Trastuzumab (final conc. ng/mL)

Figure 1. Example of Trastuzumab calibration curve using
Firefly Luciferase substrate Dual-Glo® Luciferase Reagent.
Values are shown as mean of triplicate ± SD and values on xaxis are given as final concentration in the wells before
addition of Dual-Glo® Luciferase Reagent.

Table 1. Suggested calibrator solution concentrations
for trastuzumab.

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

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APPLICATION NOTE
ProtocolAssay preparation and incubation
1. Design a plate layout.
2. Dilute calibrators, controls and samples to fall within the expected in assay values (= finalconcentration) of 0-2000 ng/mL.
3. Add 40 µL calibrators, controls and samples in duplicate to assigned wells.
4. Thaw the vial of ADCC Effector (V) Assay Ready Cells and the vials of HER2 (+) Target
Assay Ready Cells and HER2 (-) Target Assay Ready Cells in a 37C water bath withgentle agitation1.
5. Mix the cell suspensions very carefully at least 10 times with a pipette in order to ensurea homogeneous distribution of cells.
6. Dilute 200 µL of the ADCC Effector (V) Assay Ready Cells and 200 µL the HER2 (+)
Target Assay Ready Cells with 3.44 mL Diluent. The total volume of the diluted ADCC
Effector (V) /Target HER2 (+) Assay Ready Cells mixture is 3.84 mL.
7. In a separate tube, dilute 50 µL of the ADCC Effector (V) Assay Ready Cells with 50 µL ofthe HER2 (-) Target Assay Ready Cells with 860 µL Diluent. The total volume of the diluted
ADCC Effector (V)/Target HER2 (-) Assay Ready Cells mixture is 960 µL.
8. Add 40 µL of the diluted cells to each well to be tested.
9. Place the lid on the plate and mix on a plate shaker at minimum of 750 rpm for 10 sec.
Alternatively, mix the cell suspensions very carefully in the wells by pipette. Insufficientmixing can cause reduced assay sensitivity.
10. Incubate for 4 hours at 37°C with 5% CO2.
Adding substrate solutions
11. Equilibrate the plate and the substrate solutions to room temperature.
12. Prepare the Firefly luciferase substrate in accordance with the supplier’s instructions andadd 80 µL per well. Mix and protect the plate from light. After 10 minutes incubation atroom temperature read in a luminometer.
13. If appropriate, prepare the Renilla luciferase substrate in accordance with the supplier’sinstructions and add 80 µL per well. Mix and protect the plate from light. After 10 minutes
incubation at room temperature read in a luminometer.

1The color of the cell suspension might vary from salmon pink to a more yellowish red. The difference in color
is due to small variations in pH of the solution and do not affect function of the cells.
Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

SwedenSwedenLABEL-DOC-0402, 3.0
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APPLICATION NOTENormalization
The reporter gene used for normalization, Renilla luciferase, is under the control of a tyrosine kinase
promoter, and is thus constitutively expressed. Unspecific effects such as serum matrix effects ordifferences in cell number can be obviated by relating the specific Firefly signal with the Renilla
normalization signal through simple division.
B

1010
0
1000
1000
01

0.
1

0.
00
01
0.
00
1
0.
01

000
010

0

10
0

10

10

1

0.
1

0.
01

0.

00
01
0.
00
1

Relative light units (FL)AC
Figure 2.

500

A: Measurement of the specific Firefly (FL) signal. Cells
were stimulated with increasing concentrations ofTrastuzumab.

400300200Effector (V) and Target HER2 (+)
Effector (V) and Target HER2 (-)

100

C: Dose response curve after normalization of thespecific Firefly signal with the according Renilla signal.
000

1000
10

10010

101

0.
10.

01

0.
001

00.
00

B: Measurement of the Renilla (RL) signal fornormalization, from the identical assay as in A.

Trastuzumab (final conc. ng/mL)

Precautions
This application note is intended for professional laboratory research use only. The data and results
originating from following the Application Note should not be used either in diagnostic procedures
or in human therapeutic applications.
Use and handle the material and instruments referenced according to the suppliers'/manufacturers'
instructions or product specifications accompanying the individual material and instruments.
Dispose of all sample specimens, infected or potentially infected material in accordance with good
microbiological practice. All such materials should be handled and disposed as though potentially
infectious.
Residues of chemicals and preparations are generally considered as biohazardous waste andshould be inactivated prior to disposal by autoclaving or using bleach. All such materials should be
disposed of in accordance with established safety procedures.

Proprietary Information

In accepting delivery of iLite® Assay Ready Cells the recipient agrees not to sub-culture these cells,attempt to sub-culture them or to give them to a third-party recipient, and only to use them directly inassays. iLite® cell-based products are covered by patents which are the property of Svar Life Science
AB and any attempt to reproduce the delivered iLite® Assay Ready Cells is an infringement of thesepatents.

Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

SwedenSwedenLABEL-DOC-0402, 3.0
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APPLICATION NOTEQUICK GUIDE
Quantification of anti-HER2 ADCC activity usingiLite® ADCC Assay Ready Cells

1Sample dilution•Equilibrate reagents and samples to room temperature – do not thaw cells andsubstrate reagents at this stage•Dilute calibrators, controls and samples.
•Add 40 µL of calibrators, controls and diluted samples to pre-assigned wells.
•Thaw a vial of ADCC Effector (V) Assay Ready Cells, a vial of HER2 (+) Target
Assay Ready Cells, and a vial of HER2 (-) Target Assay Ready Cells in a 37C waterbath. Mix the cell suspensions with a pipette in order to ensure a homogeneous
distribution of cells.
•Prepare diluted cell mix of ADCC Effector (V)/Target HER2 (+) Assay Ready Cells,and ADCC Effector (V)/Target HER2 (-) Assay Ready Cells respectively.
•Add 40 µL diluted cell mixtures to each well and mix.

•Incubate at 37 °C with 5% CO2 for 4 hours.

2Incubation
6h3•Equilibrate the plate to room temperature•Prepare the Firefly luciferase substrate according to the supplier's instructions
and add 80 µL per well. Mix. Protect the plate from light. After 10 min incubation
read in a luminometer.
•If appropriate, prepare the Renilla luciferase substrate according to thesupplier's instructions and add 80 µL per well. Mix. Protect the plate from light.
After 10 min incubation read in a luminometer.

Read plate

Troubleshooting and FAQ
Please consult the Svar Life Science website www.svarlifescience.com

References
1. Weiner GJ. Building better monoclonal antibody-based therapeutics. Nat Rev Cancer 15(6):361-70 (2015).
2. Advani PP, Ballman KV, Dockter TJ, Colon-Otero G, Perez EA. Long-Term Cardiac Safety
Analysis of NCCTG N9831 (Alliance) Adjuvant Trastuzumab Trial. J Clin Oncol, 34(6):581-7,(2016)
3. Vu T., & Claret, F.X. Trastuzumab: Updated Mechanisms of Action and Resistance in BreastCancer. Frontiers in Oncology 2:62 (2012).
4. Rugo HS, Linton KM, Cervi P, Rosenberg JA, Jacobs I. A clinician’s guide to biosimilars inoncology, Cancer Treat Rev, 46:73-79, (2016)
Svar Life Science AB
Lundavägen 151

P.O. Box 50117

info@svarlifescience.com

SE-212 24 Malmö

SE-202 11 Malmö+46 40 53 76 00

SwedenSwedenLABEL-DOC-0402, 3.0
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