Ultra low igg serum

Adherent target cells (CHO-mCD39) were seeded in a 96-well plate (8 × 10³ cells/100 μL/well) and incubated for 24 hours. Cells were then washed twice with ADCC assay buffer (DMEM or RPMI 1640 medium supplemented with 4% Ultra-Low IgG serum (Thermo Fisher Scientific) and incubated with serially diluted mAbs (CTRL-m1, GoInVivo Purified Mouse IgG1κ control antibody, BioLegend), WT 5F2-m1, WT 5F2-m2c, and Afuc 5F2-m2c αmCD39 antibodies) for 30 minutes at 37°C. Effector cells (NFAT-luc⁺ mFcγRIV Jurkat cells, 3 × 10⁶ cells/mL) were then added to the wells and the mixture (E:T = 1:6) was incubated for 6 hours at 37°C. Bio-Glo Luciferase Assay Reagent (Promega) was finally added into wells and luminescence values were read at 30 minutes using a Synergy Neo2 Multi-Mode Reader (BioTeK Instruments Inc.). ADCC activity was indicated by an increase in luciferase activity over the background.

HCT116/HiBiT/pMIF target cancer cells were seeded at 1 × 10⁴ cells/well in 50 μL/well RPMI1640 medium supplemented with 5% ultralow IgG serum (Thermo Fisher Scientific) in 96-well flat-bottom white plates (Costar). On the next day, 50 μL/well of antibody dilutions (0.06–100 nmol/L), followed by 50 μL/well of freshly thawed PBMCs from healthy volunteers were added (5 × 10⁵ cells/well). This results in a ratio of PBMCs to target cells of 50:1. After overnight incubation of target cells with mAbs and PBMCs, target cell lysis was measured by quantifying the release of a HiBiT-tagged HaloTag protein. For this, 10 μL/well of the freshly prepared Nano-Glo HiBiT Extracellular Reagent (Promega, # N2421) was added. After incubation for 3 minutes, luminescence was measured on a Tecan Infinite M200 PRO (RRID:SCR_019033) microplate reader. Results are displayed in RLU (mean and range of n = 2). EC₅₀ values were calculated as described earlier. The experiment was repeated using PBMCs from 3 different healthy donors.

PBMCs (5 × 10⁵ cells/well) were incubated in 96-well plates with ON203 or C0008 [70, 7, 0.7, 0.07, 0 nmol/L (i.e., medium)] in 150 μL/well RPMI1640 medium supplemented with 5% ultra-low IgG serum (Thermo Fisher Scientific) for 24 hours in the presence or in the absence of HCT116/pMIF target cells (1 × 10⁴ cells/well). Cytokine release was assessed from supernatants using LegendPlex cytometric bead assays (BioLegend, RRID:SCR_001134), for human IL6, MCP-1, TNFα, IFNγ, and IL2 according to the manufacturer's protocol. Samples were read on the CytoFlex-S cytometer (Beckman Coulter, RRID:SCR_019627), and data were analyzed using LegendPlex analysis software Version 8.0 (BioLegend, RRID:SCR_001134).