M1000 microplate reader

Example 15

• • a. To test the potency of a LALA mutation in the constant part of an IgG1 antibody in diminishing antibody-mediated effector function, e.g. ADCC, a Jurkat effector cell reporter cell line based assay (Promega ADCC Bioassay, #G701A) has been applied according to manufacturer's instruction and using HEK-Blue-CD40L™ cells as target cells. 5000 HEK-Blue-CD40L™ cells were seeded per well in a white flat-bottom 384-well assay plate in 25 μl DMEM+10% FCS and incubated 20 h at 37° C., 5% CO₂. Medium was replaced with 8 μl RPMI medium containing 4% low-IgG FCS before 4000 effector cells per well were added in 8 μl of the same medium. Finally, CP-870,893 anti-CD40 antibodies, either containing an IgG1 or an IgG1-LALA Fc-part, were added in 8 μl medium at concentrations ranging from 10000 to 0.002 ng/ml. The plate was incubated for 6 h at 37° C., 5% CO₂. Effector cell luciferase activity was measured using BioGlo Luciferase assay reagents (Promega) according to the manufacturer's instructions. Luminescence was read using a Tecan M1000 microplate reader. Fold of induction was calculated with the formula RLU (antibody treatment−background)/RLU (vehicle−background). Fitting curves were obtained by using Excel (Microsoft) and XLfit (IDBS). FIG. 20 demonstrates that the LALA mutation in IgG1 abrogates Fc-receptor mediated signaling in effector cells.

Example 4

Binding of humanized anti-CD40 IgG1-LALA monoclonal antibodies to cynomolgus monkey-CD40 protein was tested in a biochemical ELISA. Recombinant cyno-CD40 protein (Acro Biosystems) was incubated in a 384-well Nunc™ MaxiSorp™ plate at a concentration of 0.5 μg/ml in PBS for one hour at room temperature. After washing three times with wash buffer (PBS, 0.1% Tween), plates were blocked with PBS, 2% BSA, 0.05% Tween for one hour at room temperature. Plates were washed again three time with wash buffer and antibodies at concentrations ranging from 500 to 0.03 ng/ml in PBS, 0.5% BSA, 0.05% Tween were incubated for one hour at room temperature. After 3 washes in wash buffer, wells were incubated with 12.5 μl of a 1:3000 dilution of anti-human peroxidase-linked, species specific F(ab)₂ Fragment from goat (AbD Serotec) in ELISA buffer for one hour at room temperature. Wells were washed six times with wash buffer and 15 μl/well TMB substrate solution (Invitrogen) were added. After 10 minutes at room temperature 15 μl Stop solution (1M HCl) were added per well and absorbance at 450 and 620 nm wavelength was measured using a Tecan M1000 microplate reader. Fitting curves and EC50 calculation were obtained by using Excel (Microsoft) and XLfit (IDBS). As shown in FIG. 4, most antibodies bind to cyno-CD40 with EC50 values between 8 and 31.8 ng/ml.

Example 21

To test the activity of IgG-1 and IgG1-LALA versions of a humanized anti-IL1R3 antibody in eliciting Fc-mediated effector cell functions such as ADCC, MAB-16-0030 was produced as an IgG1 or IgG1-LALA antibody. hIL1R3 expressing target cells SK-MEL-30 cells were seeded in a 384-well tissue-culture treated plate at a density of 2500 cells/well in 25 μl RPMI medium containing 10% FCS. 24 h after seeding, 4000 effector cells/well (ADCC Bioassay Effector cells, Jurkat, Promega Cat. #G701A) were added in RPMI medium containing 4% low-IgG-FCS. Antibodies were then added to final concentrations ranging from 10000 to 0.002 ng/ml and the plate was incubated for 6 hours at 37° C. and 5% CO₂. Activation of NF-kB signaling in luciferase gene reporter Jurkat cells was measured according to manufacturer's instructions (Bio-Glo Luciferase Assay) and using a Tecan M1000 microplate reader. “Fold of induction” values represent RLU (antibody treated−background)/RLU (no antibody control−background). Fitting curves and EC50 calculation were obtained by using Excel (Microsoft) and XLfit (IDBS). As shown in FIG. 24, only the IgG1-version of MAB-16-0030 induces NF-kB signaling in effector Jurkat reporter cells, while effector cell activation with the IgG1-LALA version is abolished.