Pd 1 pd l1 blockade bioassay

The PD-1/PD-L1 blockade bioassay (Promega, J1252) was performed to evaluate the PD-1/PD-L1 binding blockade activity of the antibodies. In brief, adherent PD-L1 aAPC/CHO-K1 cells (4×10⁵ cells/ml, 100 µl each well) were added to the inner 60 wells of a 96-well flat, white bottom assay plate. The plates were incubated at 37°C in 5% CO₂ overnight (16–20 h). On the assay day, the medium was aspirated from the inner 60-well plates. The assay plate was supplemented with serial dilutions of antibody samples (40 µl/well) and Jurkat/PD-1 effector cells (1.25×10⁶ cells/ml, 40 µl/well) and then incubated at 37°C in 5% CO₂ for 6 h. The assay plate was allowed to stand at room temperature for 15 min, and then the Bio-Glo substrate reagent (Promega, G7940) was added to 80 µl of each well and incubated in the dark at room temperature for 5 min. Luminescence signals were measured using the Cytation 5 cell imaging multimode reader and reported as a relative light unit (RLU). The IC₅₀ was calculated using GraphPad Prism software.

Cell viability was measured using CellTiter-Glo or LDH-Glo (G7570, J2380, Promega). Briefly, CD22 positive or negative cells were plated into 96-wells, allowing attachment and growth for 24 hr, then triplicate wells were treated with DDCs, naked antibodies, free drugs, or DDCs plus competitor antibodies. Three to five days later, when untreated control wells were 70 to 90% confluent, reagent was added to the plates according to the supplier’s instructions. Wells treated identically but wells without cells were used to subtract background. Fluorescence (ex: 570 nm, Em: 585 nm) was measured using a CLARIOstar microplate reader (BMG Labtech) and data analyzed using GraphPad Prism 8.0.1 software. Significance was tested using one-way ANOVA, followed by the Tukey’s multiple post hoc test. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05 between the indicated groups. PD-L1 neutralization was done in a PD-1/PD-L1 Blockade Bioassay (J1250, Promega).

The activity of the masked antagonists was analyzed using a PD-1/PD-L1 Blockade Bioassay (Promega, J1250) according to a modified manufacturer’s protocol as described previously(32 (link)). Briefly, PD-L1 aAPC/CHO-K1 cells were seeded into a 96-well assay plate and incubated for 16 h. For all cleaved masked antagonists, each construct (4 μM) was incubated with 75% molar equivalent TEV in assay buffer at 37°C for 16 h. The non-cleaved masked complexes (4 μM) were incubated at 4°C for 16 h. Masked complexes ± TEV (either at 10 and 100 nM or 1:2.5 serial dilutions from 1.3 nM to 2 μM – for screening or for the full assay, respectively) were added to the plates followed by seeding PD-1 effector cells. After co-culture for 6 h, the Bio-Glo^™ reagent was added. The luminescence signal was measured using a CLARIOstar Plus microplate reader (BMG Labtech). The full assay was performed twice either in duplicate or triplicate (technical replicates) and relative luminescence units (RLU) were plot against masked complexes concentration. The data from each experiment were analyzed using the non-linear fit in Prism Version 9.4.1 with all samples constrained to the same top and bottom value (as determined for NoMA without constraints). The data across experiments were then averaged.