Fig. 1

Characterization of high-affinity SIRPα variants. A Table of engineered SIRPα variants sequences and affinity measurements. The sequence of wild-type SIRPα (SIRPα WT) and the position of the mutated amino acids are illustrated in the table. The blue shading indicates the final selected type of SIRPα variant. B Representative SPR sensorgram of SIRPα WT binding CD47. RU = response units. C Representative SPR sensorgram of selected high-affinity SIRPα variant (V5) binding CD47. RU = response units. D Titration curves of SIRPα WT (brown) and high-affinity SIRPα variants (SIRPα variant, blue) binding to erythrocytes. E Dose–response curves of CD47 antagonism on erythrocytes with SIRPα WT (brown), CD47 antibody clone B6H12 (anti-CD47, red), and V5 SIRPα variant (SIRPα variant, blue). Cells were stained with different concentrations of CD47 blocking agents in competition with Alexa Fluor 594-conjugated wild-type SIRPα tetramer. F Fitting curves of SIRPα variant (blue) and SIRPα WT (brown) binding to mouse erythrocytes. Binding assays of biotinylated SIRPα WT and variants were performed with Alexa Fluor 594-conjugated streptavidin. G Mouse CD47 blocking assay. SIRPα variant (blue) and wild-type mouse SIRPα (mSIRPα WT, brown) block Alexa Fluor 594-conjugated wild-type mouse SIRPα tetramers binding to mouse CD47 displayed on the surface of yeast. All the data are presented as the mean ± SD