Co-incubation of Acasunlimab with PD-1-NF-AT-Jurkat and CD3L-huPD-L1-CHO-K cells and incubated for 6 hours. Bright-Lite was used to detect the fluorescent signal. As shown in fig, Acasunlimab was able to block the PD-1/PD-L1 signaling pathway, and the EC50 was 1.875 nM.

Co-incubation of Acasunlimab with 4-1BB-NF-κB-Jurkat cells, then with the addition of huPD-L1-CHO-K cells for 6 hours. Bright-Lite was used to detect the fluorescent signal. As shown in fig, Acasunlimab was able to activate the NF-κB signaling pathway, and the EC50 was 0.341 nM.

Acasunlimab bound to huPD-L1-CHO-K cells, and then rebounded to fluorescent secondary antibodies(Anti-human IgG, Fcγ PE) , and test by flow cytometry. As shown in fig, Acasunlimab bound to huPD-L1-CHO-K cells, and the EC50 was 1.153 nM.

Acasunlimab bound to hu4-1BB-CHO-K cells, and then rebounded to fluorescent secondary antibodies(Anti-human IgG, Fcγ PE) , and test by flow cytometry. As shown in fig, Acasunlimab bound to hu4-1BB-CHO-K cells, and the EC50 was 3.592 nM.

To measure the binding ability of Acasunlimab in hu-PD-L1-His. Acasunlimab bound to PD-L1 protein, and then rebounded to secondary antibodies(Anti-human-IgG-Fc-HRP) , and read OD450. As shown in fig, Acasunlimab bound to hu-PD-L1-His, and the EC50 was 0.042 nM.

To measure the binding ability of Acasunlimab in hu-4-1BB-His. Acasunlimab bound to 4-1BB protein, and then rebounded to secondary antibodies(Anti-human-IgG-Fc-HRP) , and read OD450. As shown in fig, Acasunlimab bound to hu-4-1BB-His, and the EC50 was 0.007 nM.

The purity of Anti-4-1BB & PD-L1 Reference Antibody (Acasunlimab) is 94.41%, determined by SEC-HPLC.

Anti-4-1BB & PD-L1 Reference Antibody (Acasunlimab) on SDS-PAGE under reducing (R) condition. The purity of the protein is greater than 95%.