Co-incubation of Yh32367 with BT474 cell and PBMCs for 4 hours, then LDH was detected to evaluate the ADCC activity of Yh32367. As shown in fig, Yh32367 has ADCC activity, and the IC50 was 0.054 nM.

Co-incubation of Yh32367 with 4-1BB-NF-κB-Jurkat cells, then with the addition of BT474 cells for 6 hours. Bright-Lite was used to detect the fluorescent signal. As shown in fig, Yh32367 was able to activate the NF-κB signaling pathway, and the EC50 was 0.355 nM.

Yh32367 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, Yh32367 bound to hu4-1BB-CHO-K cells, and the EC50 was 5.701 nM.

Yh32367 bound to BT474 cells, and then rebounded to fluorescent secondary antibodies(Anti-Human IgG, Fcγ PE) , and test by flow cytometry. As shown in fig, Yh32367 bound to BT474 cells, and the EC50 was 1.889 nM.

Yh32367 bound to Her2 protein, and then rebounded to secondary antibodies(Anti-Human-IgG-Fc-HRP) , and read OD450. As shown in fig, Yh32367 bound to hu-Her2-His, and the EC50 was 0.009 nM.

Yh32367 bound to Her2 protein, and then rebounded to secondary antibodies(Anti-Human-IgG-Fc-HRP) , and read OD450. As shown in fig, Yh32367 bound to hu-Her2-His, and the EC50 was 0.009 nM.

Yh32367 bound to 4-1BB protein, and then rebounded to secondary antibodies(Anti-Human-IgG-Fc-HRP) , and read OD450. As shown in fig, Yh32367 bound to hu-4-1BB-His, and the EC50 was 0.007 nM.

The purity of Anti-4-1BB & TPBG/5t4 Reference Antibody (Apv-527) is 98.99%, determined by SEC-HPLC.

Anti-4-1BB & TPBG/5t4 Reference Antibody (Apv-527) on SDS-PAGE under reducing (R) condition. The purity of the protein is greater than 95%.