Figure 1.

BsAb CD47xEGFR-IgG1 selectively and simultaneously binds to EGFR and CD47. Dose-dependent binding of CD47xEGFR-IgG1 to (a) CHO.EGFR or (b) CHO.CD47 cells vs. parental CHO cells. (c) Binding of CD47xEGFR-IgG1 or CD47xMock-IgG1 (1 µg/ml) to a panel of EGFR^pos and EGFR^neg CD47-expressing cell lines. (d) Binding of CD47xEGFR-IgG1 (1 µg/ml) to (GFP-labeled) CHO.CD47 cells and (DiD-labeled) parental A431 or A431.CD47KO cells in the presence or absence of excess CD47-blocking antibody B6H12 and/or EGFR-blocking antibody mAb 425 (both 10 µg/ml). The percentage of DiD/GFP double positive events upon incubation with indicated antibodies is shown. (e) Competitive binding assay in which APC-conjugated CD47 antibody (B6H12) competed with increasing doses (0.01–10 µg/ml) of bsAb CD47xEGFR-IgG1 (white squares), CD47xMock-IgG1 (black triangles) or MockxMock-IgG1 (white dots) for binding to A431 cells. (f) Competitive binding assay, as in E, in which A431 cells were pretreated with excess amounts of mAb 425 or isotype control IgG2a (both 10 µg/ml) prior to addition of CD47xEGFR-IgG1 (1 µg/ml), where indicated. (g) In vitro binding of CD47xEGFR-IgG1 and B6H12-hIgG1 to RBC present in whole blood. (h) Binding assay of CD47xEGFR-IgG1 (20 µg/ml) to DiO-labeled NCI-H292 cancer cells in the presence of increasing concentrations of whole blood. Of note, RBC in whole blood form an abundant “antigen-sink” for CD47-antibodies. All binding experiments were analyzed by flow cytometry. All graphs represent mean ± SD. Statistical analysis in F was performed using one-way ANOVA followed by a Tukey post-hoc test (*p < .05, **p < .01, ***p < .001, ****p < .0001, ns not significant)