Adcc bioassay effector cells

Example 9

ADCC Reporter Bioassay

2.5×10⁴ HCC1428 cells in 100 μL culture medium/well were incubated in 96-well cell culture plate (Greiner, Cat #655083-1) at 37° C. in a humidified 5% CO₂ incubator (NUAIRE, Cat #NU-4750) overnight. Culture medium was replaced with 2× serial dilution of 37.5 μL OBI-898 from 20 to 0.000305 μm/mL in ADCC Assay Buffer (Promega, Cat #G708A, G711A). In addition, background wells which contained no antibody were included. In each well, 37.5 μL of 7.5×10⁴ ADCC Bioassay Effector cells (Promega, Cat #G701A) were added. Induction was performed for six hours and 75 μL of Bio-Glo Luciferase Assay Reagent (Promega, Cat #G719A, G720A) was added. After 15 minutes, luminescence (RLU, relative light unit) was determined using a microplate luminometer, SpectraMax L (Molecular Devices, Sunnyvale, Calif.).

Luminescence induction of fold changes was calculated by the ratio of relative light unit (RLU) (induced) to RLU (no antibody control). EC₅₀ was determined by plotting x (concentration in μg/mL)−y (the induction of fold change) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software.

According to FIG. 15, The EC₅₀ response of c1J1s, H4-16K2, H4-16K2 N56Q, H4-16K2 N56S, H4-16K2 N58Y and H4-16K2 K3T/N56Q were 987.2, 50.7, 9.7, 9.2, 21.2 and 8.3 μg/mL, respectively. It showed that H4-16K2 K3T/N56Q clone has best ADCC activity.

Example 9

ADCC Reporter Bioassay

2.5×10⁴ HCC1428 cells in 100 μL culture medium/well were incubated in 96-well cell culture plate (Greiner, Cat #655083-1) at 37° C. in a humidified 5% CO₂ incubator (NUAIRE, Cat #NU-4750) overnight. Culture medium was replaced with 2× serial dilution of 37.5 μL OBI-898 from 20 to 0.000305 μg/mL in ADCC Assay Buffer (Promega, Cat #G708A, G711A). In addition, background wells which contained no antibody were included. In each well, 37.5 μL of 7.5×10⁴ ADCC Bioassay Effector cells (Promega, Cat #G701A) were added. Induction was performed for six hours and 75 μL of Bio-Glo Luciferase Assay Reagent (Promega, Cat #G719A, G720A) was added. After 15 minutes, luminescence (RLU, relative light unit) was determined using a microplate luminometer, SpectraMax L (Molecular Devices, Sunnyvale, Calif.).

Luminescence induction of fold changes was calculated by the ratio of relative light unit (RLU) (induced) to RLU (no antibody control). EC₅₀ was determined by plotting x (concentration in μg/mL)−y (the induction of fold change) and fitting the data in a 4PL nonlinear regression model by PRISM 6 Software.

According to FIG. 15, The EC₅₀ response of c1J1s, H4-16K2, H4-16K2 N56Q, H4-16K2 N56S, H4-16K2 N58Y and H4-16K2 K3T/N56Q were 987.2, 50.7, 9.7, 9.2, 21.2 and 8.3 μg/mL, respectively. It showed that H4-16K2 K3T/N56Q clone has best ADCC activity.