FIGURE 2.

AL008 induces the internalization and degradation of SIRPα on human macrophages. (A) BWZ-huSIRPα overexpressing cells were incubated in the presence of AL008 or with CD47-blocking anti-SIRPα Abs (i.e., KWAR23, 18D5), followed by incubation with allophycocyanin-labeled human CD47 tetramers. The percentage of CD47 tetramer binding was measured and normalized to Ab control condition. (B) A receptor internalization assay was performed on monocyte-derived macrophages treated overnight with increasing concentrations of the indicated Abs. A competition assay was performed under similar conditions, except macrophages were treated with test Abs at 4°C for 30 min. Cell surface SIRPα was detected by flow cytometry with a fluorescent anti-human SIRPα Ab that does not compete with test Ab binding. Relative expression levels of SIRPα were normalized to the isotype-treated cells. Data are means ± SD. (C) Macrophages from five donors were pretreated with AL008 and then treated with vehicle, PMA, or LPS (left panel). The supernatant was collected and levels of soluble SIRPα were determined (right panel). Data are means ± SEM. (D) The protease inhibitor batimastat prevents the PMA- and LPS-induced increase in soluble SIRPα. Data are means ± SEM. Soluble SIRPα values from isotype-treated vehicle control cells were normalized for each donor. (E) Western blot analysis reveals AL008-mediated degradation of SIRPα. (F) Macrophages that are homozygous for the v1 variant of SIRPα (left) or are expressing a non-v1 variant of SIRPα (right) were treated overnight with increasing concentrations of AL008, CD47-blocking anti-SIRPα Abs, or isotype control. Cells were stained with allophycocyanin-labeled human CD47 tetramers. Relative CD47 binding was normalized to isotype-treated cells. Isotype control shown is human IgG1 NSLF. Data are means ± SD. For (C) and (D), a one-way ANOVA was performed followed by Holm–Sidak’s method of multiple comparisons. *p < 0.05, ***p < 0.001, ****p < 0.0001. ns, not significant.