2.4. CDC and ADCC assays

ADCC assay

Materials and Methods

1. Reagents
RPMI1640: Gibco (Thermo Fisher Scientific K.K.), Cat. No. 11875-093, 11875-085 
RPMI1640, HEPES: Gibco (Thermo Fisher Scientific K.K.), Cat. No. 22400-089 
RPMI1640 (no phenol red): Gibco (Thermo Fisher Scientific K.K.), Cat. No. 11835-030 
Fetal bovine serum (FBS): Hyclone (GE Healthcare), Cat. No. SH30406.02 
0.4 % Trypan blue solution: Wako Pure Chemical Industries, Ltd., Cat. No. 207-17081 
DPBS: Gibco (Thermo Fisher Scientific K.K.), Cat. No. 14190-144
ADCC Reporter Bioassay, Core kit: Promega Corporation, Cat No. G7010
<Components>
ADCC Bioassay Effector Cells, RPMI 1640 Medium, Low IgG Serum, Bio-Glo™ Luciferase Assay Buffer, Bio-Glo™ Luciferase Assay Substrate

2. Test substances

2.1 Test substance 1 JR-141 (8.38 mg/mL)

2.2 Test substance 2 humanized anti-TfR mAb (3.30 mg/mL)

3. Target cells

Human T lymphoma cell line, CCRF-CEM, was purchased from Japanese Collection of Research Bioresources (JCRB) cell bank (JCRB No. JCRB9023), and used in this study. Human myelogenous leukemia cell line, K562, was purchased from Riken BioResource Center cell bank (No. RCB0027), and used in this study.

Human human erythroblast cell line, HEL92.1.7, was purchased from American Type Culture Collection (ATCC) cell bank (No. ATCC TIB-180), and used in this study.

4. Effector Cells

Genetically engineered Jurkat cells stably expressing the FcγIIIa receptor, V158 (high affinity) variant, and an NFAT (nuclear factor of activated T-cells) response element driving expression of firefly luciferase was used as effector cells.

5. Cell culture

CCRF-CEM and K562 were cultured in RPMI1640 containing 10% heat-inactivated FBS and 2 mM L-glutamine, and incubated at 37°C in a humidified atmosphere of 5% CO₂. HEL92.1.7 were cultured in RPMI1640 containing 10% heat-inactivated FBS, 25 mM HEPES and 2 mM L-glutamine, and incubated at 37°C in a humidified atmosphere of 5% CO₂.

6. Preparation of serial dilutions of test substances

Humanized anti-hTfR mAb and JR-141 were serially diluted in RPMI1640 medium (containing 10% low-IgG FBS) to the final concentration of 0.0274 to 60.0 μg/mL and 0.0549 to 360 μg/mL, respectively (corresponding to 0.188 ~ 822 μmol/L and 0.207 ~ 1358 μmol/L, respectively). The dilution factor at each step was three.

7. Preparation of effector cells

The vial of ADCC Bioassay Effector Cells was thawed in a 37°C water bath until cells were just thawed. After gently mixing, 630 µL of the cell suspension was transferred to 15 mL tube containing 3.6 mL of RPMI1640 medium (containing 10% low-IgG FBS), and mixed well.

8. ADCC assay

25 μL of target cell suspension (4 × 10⁵cells/well) were seeded into 96-well black microplate in RPMI1640 medium (containing 10% low-IgG FBS) (1.0 x 10⁴ cells/well). Then, 25 μL of the test substances were added to the wells (duplicate), and mixed for 30 sec with microplate mixer. Subsequently, 25 μL of effector cell suspension was added to the well. Untreated control wells (without test substances) and background control wells (without the test substances and cells) were also made. Then, the cells were incubated at 37°C in a humidified atmosphere of 5% CO₂ for 6hrs.

Table 1. Final concentration of test substances in micro plate.

After incubation period,25 µL of Bio-Glo Luciferase Assay Reagent was added to the test well and mixed for 30 sec with microplate mixer. Then, the 96 well plate was incubated at room temperature for 15 mins to develop luminescent material. After incubation period, resulting luminescence was measure using a plate reader with glow-type

luminescence read capabilities (GloMax96 Microplate Luminometer).

9. Data processing

The average luminescence intensity of background control wells was subtracted from the luminescence intensities of the test substances and untreated control wells. The average luminescence intensities were calculated from corrected experimental intensities. Dose-response curve was constructed by plotting the values of percent cytotoxicity on the vertical (Y) axis against nominal concentrations of test substances on the horizontal (X) axis. The horizontal axis was showed on logarithmic scale.

CDC assay

Materials and Methods
1. Reagents
RPMI1640: Gibco (Thermo Fisher Scientific K.K.), Cat. No. 11875-093, 11875-085, 22400-089
RPMI1640 (no phenol red): Gibco (Thermo Fisher Scientific K.K.), Cat. No. 11835-030 
Fetal bovine serum (FBS): HyClone (GE Healthcare), Cat. No. SH30406.02
0.4 % Trypan blue solution: Wako Pure Chemical Industries, Ltd., Cat. No. 207-17081 
DPBS: Gibco (Thermo Fisher Scientific K.K.), Cat. No. 14190-144
Normal Human Serum Complement: Quidel Corporation, Cat. No. A113 
Digitonin, high purity: Merck KGaA, Cat. No.300410-250MG
Dimethyl sulfoxide (DMSO): Sigma-Aldrich Co., LLC., Cat. No. D8418-250ML 
CytoTox-Fluor Cytotoxicity Assay Kit: Promega Corporation, Cat No. G9261
<Components>
bis-AAF-R110 Substrate, Assay Buffe
 

2. Test substances
2.1. Test substance 1 JR-141 (8.38 mg/mL)
2.2. Test substance 2 humanized anti-TfR mAb (3.30 mg/mL)
 

3. Target cells
Human T lymphoma cell line, CCRF-CEM, was purchased from Japanese Collection of Research Bioresources (JCRB) cell bank (JCRB No. JCRB9023), and used in this study. Human myelogenous leukemia cell line, K562, was purchased from Riken BioResource Center cell bank (No. RCB0027), and used in this study.
Human human erythroblast cell line, HEL92.1.7, was purchased from American Type Culture Collection (ATCC) cell bank (No. ATCC TIB-180), and used in this study.
 

4. Normal human serum complement
Normal human serum complement was purchased from Quidel Corporation (San Diego, USA), stored in aliquots at -80°C. Repeated freeze-thaw cycles were avoided. Normal human serum complement was thawed and diluted 1:2.5 with RPMI1640 medium (no phenol red) immediately before using. (40% human complement)
 

5. Cell culture
CCRF-CEM and K562 were cultured in RPMI1640 containing 10% heat-inactivated FBS and 2 mM L-glutamine, and incubated at 37°C in a humidified atmosphere of 5% CO₂. HEL92.1.7 were cultured in RPMI1640 containing 10% heat-inactivated FBS, 25 mM HEPES and 2 mM L-glutamine, and incubated at 37°C in a humidified atmosphere of 5% CO₂.
 

6. Preparation of serial dilutions of test substances
JR-141 and humanized anti-TfR mAb were serially diluted in RPMI1640 medium to the final concentration of 0.0977 to 400 µg/mL and 0.0537 to 220 µg/mL, respectively (corresponding to 0.367 ~ 1500 nmol/L). The dilution factor at each step was four.
 

7. CDC assay
25 µL of target cells (1.0 x 10⁶ cells/mL) were seeded into 96-well black microplate in RPMI1640 medium (5.0 x 10⁴ cells/well). Then, 25 µL of the test substances were added to the wells (triplicate), and mixed for 30 sec with microplate mixer. Subsequently, 25 µL of 40% human complement was added to the well, and mixed for 30 sec with microplate mixer. Untreated control wells, positive control wells (without test substances) and background control wells (without the test substances and cells) were also made. Then, the cells were incubated at 37°C in a humidified atmosphere of 5% CO₂ for 4hrs.
 

Table 1. Final concentration of test substances and human complement in micro plate.

After incubation period, 1 µL of 20 mg/mL digitonin was added to the positive control (maximum cell lysis) well and mixed for 30 sec with microplate mixer. Subsequently, 100 µL of CytoTox-FluorTM Cytotoxicity Assay Reagent (Promega Corporation, Fitchburg, Wisconsin.) was added to the well and mixed for 30 sec with microplate mixer. Then, the 96 well plate was incubated at 37°C for 1hr to develop fluorescent material. After incubation period, measured resulting fluorescence using microplate spectrofluorometer (Ex: 485 nm/ Em: 520 nm).
 

8. Data processing
The average fluorescence intensity of background control wells was subtracted from the fluorescence intensities of the test substances and untreated control wells. The average fluorescence intensities were calculated from corrected experimental intensities. The percent cytotoxicity was calculated using the equation below. Dose-response curve was constructed by plotting the values of percent cytotoxicity on the vertical (Y) axis against nominal concentrations of test substances (nmol/L) on the horizontal (X) axis. The horizontal axis was showed on logarithmic scale.