Data acquisition 6

Bio-layer Interferometry (BLI) analyses of the interaction between recombinant PD-L1 trap and PD-L1 or PD-L2 were performed on a fortéBIO Octet RED96 system. Assays were run at 30°C on Greiner Bio One black 96- well microplates. To measure the interaction between PD-L1 with the trimeric trap, AHC or SAX biosensors (Pall fortéBIO Corp.) were used to immobilize PD-L1 tagged with Fc or biotin. Purified trimeric PD-L1 trap was prepared in an assay buffer (1×PBS, 0.002% Tween 20, pH 7.4) and applied to a 96-well microplate in column arrangement. Various concentrations of trap (0–500 nM) were used to test the binding. To study the disruption of preformed PD-1/PD-L1 complex, PD-L1 immobilized on the SAX biosensor was first saturated with 200 nM of PD-1, followed by incubation with a mixture of 200 nM PD-1 with or without trimeric PD-L1 trap. Assays were run in triplicate and all data were acquired and analyzed with fortéBIO Data Acquisition 6.4 software. Data processing was performed by averaging the reference biosensors, applying Savitzky-Golay filtering, and fitting binding curves. All binding kinetics for the PD-L1 trap are reported by Miao et al. (publication pending). All binding kinetics for the CXCL12 trap are reported by Goodwin et al⁹ .

Biolayer interferometry analyses of the interaction between recombinant PD-L1 trap and PD-L1 or PD-L2 were performed on a fortéBIO Octet RED96 system. Assays were run at 30 °C on Greiner Bio One black 96-well microplates. To measure the interaction between PD-L1 with the trimeric trap, AHC or SAX biosensors (Pall fortéBIO Corp.) were used to immobilize PD-L1 tagged with Fc or biotin. Purified trimeric PD-L1 trap was prepared in an assay buffer (1× PBS, 0.002% Tween 20, pH 7.4) and applied to a 96-well microplate in column arrangement. Various concentrations of trap (0–500 nM) were used to test the binding. To study the disruption of preformed PD-1/PD-L1 complex, PD-L1 immobilized on the SAX biosensor was first saturated with 200 nM of PD-1, followed by incubation with a mixture of 200 nM PD-1 with or without trimeric PD-L1 trap. Assays were run in triplicate, and all data were acquired and analyzed with fortéBIO Data Acquisition 6.4 software. Data processing was performed by averaging the reference biosensors, applying Savitzky-Golay filtering, and fitting binding curves.

BLI analyses of the monomeric and heterodimeric EGFR and HER2-binding domains were performed on a Octet QK system (FortéBio LLC., Menlo Park, CA) against recombinant EGFR-Fc and HER2-Fc (AcroBiosystems, Newark, NJ) [17 (link), 23 (link)] using 96-well microplates (Greiner Bio-One) at 30°C. Streptavidin biosensors (FortéBio) were used to immobilize concentrations of biotinylated ErbB-binding domains and samples resuspended in an assay buffer (1× PBS, 1% BSA, 0.05% Tween 20, pH 7.4) were applied to the 96-well microplate. Assays run in triplicate were acquired and analyzed on the FortéBio Data Acquisition 6.4 software. Savitzky-Golay filtering was applied to the averaged reference biosensors and then globally fitted at a 1:1 model.