Anti his apc

OX40L (NM_003326.5) and 4-1BBL (NM_009404.3) sequences were fused with his-tag and then cloned into pLVX-EF1a-IRES-Puro (Addgene, 85132). The neo-2/15 sequence was fused with Flag-tag and cloned into pLVX-IRES-ZsGreen1 (Fenghui, BR021). The lentivirus was produced by co-transfection with the packaging plasmid psPAX2 and pMD2.G into lenti-X-293 cells cultured in DMEM medium with 10% FBS. The K562 or NK-92 were seeded into 24-well plates at 1×10⁵ cells/well and then infected at MOI=10. The cells were harvested 7 days post-infection and subsequently sorted by flow cytometry, resulting in GFP-positive or His-positive monoclonal cell lines. Anti-Flag-PE and anti-His-APC were obtained from BioLegend. NK92-based feeder cells were acclimatized in AIM-V medium after establishment.

Jurkat cells stably over-expressing hPD-1 (Jurkat-hPD-1) were engineered and used to examine the PD-1/PD-L1 blocking activity of CBPA with flow cytometry at the cell level. Different pre-incubated solutions was added into five tubes (tube 1: 100 µL PBS as the negative control; tube 2: 50 µL commercially available recombinant hPD-L1-His protein (10 ug/mL, 10084-H08H, Sino Biological) + 50 µL PBS as the positive control; tube 3: 50 µL PD-L1-His protein + 50 µL 40 µM inhibitor 1 compound (BMS-1 compound in WO 2015/034820A1 patent [19 ], Selleck, S7911); tubes 4 to 5: 50 µL PD-L1-His protein + 50 µL 200 µM or 40 µM CBPA compound) and incubated for 30 min at 4 °C. Meanwhile, cells were harvested, washed with PBS, and re-suspended in pre-cooling PBS at a concentration of 4 × 10⁶ cells/mL. Then, 100 µL of cells was added to each of the above tubes, and they were mixed gently and co-incubated on ice for an additional 60 min. Subsequently, cells were harvested, washed twice with PBS, and stained with Anti-His-APC (BioLegend) in buffer for 20 min in the dark on ice. Stained cells were washed once and measured on a flow cytometer (CytoFLEX S, Beckman), and the data were analyzed by using FlowJo software (TreeStar, Ashland, OR, USA). We calculated the blocking efficiency (%) with the formula: 100% × (1 − positive rate _treatment/positive rate _{positive control}).