Figure 1.

Pep-20 binds to CD47 and blocks the CD47/SIRPα interaction. (A, B) Dose response curves of pep-20 binding to CD47. Binding assay of pep-20 to human (A) or mouse (B) CD47-IgV-Domain protein was examined by the MST. The human or mouse SIRPα protein served as positive control. (C, D) Dose response curves of pep-20 interfering CD47/SIRPα interaction. Flow cytometry analysis of human (C) or mouse (D) CD47-IgV-Domain-hIg fusion protein binding to CHO stably expressing human or mouse SIRPα cells in the presence of pep-20 with varying gradient concentrations. The data represented as the mean fluorescence intensity normalized to the maximum binding. (E to J) Phagocytosis assays were performed by co-culture of tumor cells with corresponding macrophages at a 1∶4 ratio in serum-free medium at 37℃ for 4 hours in low-attachment 96-well plates. GFP⁺ MCF7 cells (E), GFP⁺ HT29 cells (F) and GFP⁺ Jurkat cells (G) were incubated with human peripheral blood-derived macrophages in the presence of 100 µM pep-20 or 20 µg/mL anti-human CD47 antibody (B6H12). CFSE⁺ CT26 cells (H), GFP⁺ MC38 cells (I) and GFP⁺ B16-OVA cells (J) were incubated with mouse bone marrow-derived macrophages in the presence of 100 µM pep-20 or 20 µg/mL anti-mouse CD47 antibody (miap301). Phosphate-buffered saline was negative control in all assays. CFSE⁺ or GFP⁺ macrophages were detected by flow cytometry. Data are represented as means±SEM. Statistical significance was determined by unpaired Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. CHO, Chinese hamster ovary; CSFE, carboxyfluorescein succinimidyl ester; GFP, green fluorescent protein; MST, microscale thermophoresis.