Figure 4. Humanized 1H9 binds to different SIRPα variants and synergizes with cetuximab to promote phagocytosis across donors expressing different SIRPα variants.

(A) ELISA binding of 1H9 to human SIRPα-V1, SIRPα-V2, and an irrelevant Fc fusion proteins under increasing concentrations as indicated. Each sample was assayed in triplicate. Data represent mean ± SD. (B) Monocytes derived from 3 human donors, which express SIRPα as V1/V1, V2/V2, and V1/V5 variants, were incubated with 1 μg/mL AF488-labeled CD47-Fc fusion protein in the absence or presence of increasing concentrations of humanized 1H9. Binding of CD47 on the cells was measured and analyzed by flow cytometry. Binding was calculated and normalized on the mean fluorescence intensity of CD47/SIRPα binding in the absence of 1H9 as 100%. (C) HT-29 tumor cells were labeled with CFSE and incubated with human monocyte–derived macrophages, which express SIRPα as V1/V1 (n = 10), V2/V2 (n = 2), and V1/V5 (n = 1) variants, in the presence of 10 μg/mL humanized 1H9 or 0.1 μg/mL cetuximab, either alone or in combination. Fold increase of phagocytosis was calculated by phagocytosis induced by the combination treatment over phagocytosis induced by cetuximab alone.