Cell based blocking assay

CHOK1-TIGIT cells stably expressing human or mouse TIGIT were established by lenti-viral infection followed by puromycin selection according to the previous report [6]. Cell based blocking assay was performed according to the previous report [30]. Briefly, PVR-Fc protein was incubated with a series dilution of small molecule compounds or control buffer on ice for 30 min, human PVR-Fc (CD5-H82F6, ACRO Biosystem) and mouse PVR-Fc (50259-M03H, Sino Biological) were used at concentrations of 10 and 40 ng/test, respectively. Then the mixture of PVR protein and compounds were incubated with CHOK1-TIGIT cells for another 30 min, followed by incubation with detection antibody anti-human IgG-PE (eBioscience) for 30 min. Cells were then washed with FACS buffer to remove the unbound fluorescent antibodies, acquired on a FACS Caliber™ (BD Biosciences) and analyzed by FlowJo. To test the specificity of candidate compounds, similar blocking assays were performed by using PD-1 (PD-L1-Fc, 10,084-H02H, Sino Biological) and CD47 (Sirpα-Fc, SIA-H52A8, ACRO Biosystem) over-expressed CHOK1 cells. Cells incubated with corresponding Fc tagged protein and anti-human IgG-PE served as positive control, while cells only incubated with anti-human Fc-PE served as negative control. The mean fluorescent intensity (MFI) was recorded for calculating the blocking efficiency as the formula: