Ligand-binding blockade assays
APC
The DiscoverX PathHunter U2OS PD-1/LAG-3 dimerization assay uses enzyme fragment complementation (EFC) technology for studying protein–protein interactions. In essence, EFC consists of a split β-galactosidase (β-gal): the smaller enzyme donor (ED) and the larger enzyme acceptor (EA) fragments that independently have no enzymatic activity. When the two fragments are forced to complement, they form an active β-gal that can hydrolyze substrate to produce a chemiluminescent signal. In this study, U2OS cells were engineered to stably co-express EA-tagged LAG-3 and ED-tagged PD-1 (U2OS PD-1/LAG-3 cells). In brief, the dimerization assay was performed according to the manufacturer’s protocol. Approximately 2 × 10The DiscoverX PathHunter Jurkat PD-1 signaling assay (SHP-2) uses the same EFC technology described earlier. Two engineered cells were employed for studying PD-1/SHP-2 signaling: Jurkat cells stably co-expressed ED-tagged PD-1 and EA-tagged SHP-2 (Jurkat PD-1/SHP-2 cells); U2OS cells stably expressed PD-L1 (U2OS PD-L1 ligand cells). In essence, activation of PD-1/SHP-2 signaling by PD-1 ligands or anti-PD1 antibody recapitulated β-gal activity to hydrolysis substrate for a chemiluminescent signal. The addition of PD-1/SHP-2 signaling antagonist, which prevented complementation of β-gal fragments, resulted in a loss of chemiluminescent signal. In brief, the signaling assay was performed according to the manufacturer’s protocol. Approximately 2 × 10PD-1/PD-L1 blockade bioassay (Promega) and luciferase reporter assay: this system uses Jurkat-PD-1LAG-3/MHC-II blockade bioassay (Promega) and luciferase reporter assay: this system uses Jurkat–LAG-3A cell-based dual reporter system was used to assess dual blockade of PD-1:PD-L1 and LAG-3:MHC-II (Promega PD-1/LAG-3 combination bioassay). Two engineered cell lines were employed for studying PD-1–LAG-3 engagement in this combination bioassay: a suspension cell line stably co-expressed PD-1 and LAG-3 (PD-1 + LAG-3 effector cells), and an adherent cell line stably co-expressed PD-L1 and MHC-II (PD-L1 + MHC-II antigen-presenting cell (APCs)). In the native state, the reporter activity in PD-1 + LAG-3 effector cells was suppressed when the cells co-ligated with PD-L1 + MHC-II APC cells and in the presence of an assay-specific MHC-II peptide (TCR-activating antigen). The reporter activity in PD-1 + LAG-3 effector cells was restored with the addition of PD-1 and/or LAG-3 antagonist, which disrupted the respective PD-1:PD-L1 and/or LAG-3:MHC-II interactions between the two engineered cells. In brief, the combination bioassay was performed according to the manufacturer’s protocol. Approximately 2 × 10