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. 2022 Sep 12;10(9):e004554. doi: 10.1136/jitc-2022-004554

Figure 1.

Figure 1

bsAb AD2×1E9 binds simultaneously with both Fab arms, is resistant to APCP-mediated epitope sequestration and exhibits increased Ab deposition. (A) Competition of fluorescently labeled Abs (AD2-AF488, 1E9-AF488, MEDI9447-AF594, bsAb AD2×1E9-AF488, 5 µg/mL) with preincubated non-labeled Abs (indicated to the left, 10 µg/mL) and analyzed by flow cytometry. Peaks shifted to the left indicate blocking of the labeled Ab. Panel (A) is representative of three independent experiments. (B, C) Increased binding by the AD2-AF488 in the presence of 1E9 and bsAb 1E9xb12 was observed, whereas binding of 1E9-AF488 was not affected by the presence of AD2 or bsAb AD2xb12, as determined by flow cytometry analysis. (D) Ab binding in the presence or absence of APCP as determined by flow cytometry analysis. Epitope recognition by anti-CD73 Abs was detected by addition of a secondary fluorescent-labeled Ab. Mean±SEM from three independent experiments is shown. Statistical difference was determined using paired t-tests. (E) Schematic representation of the epitope interactions. (F) Flow cytometry analysis of Ab binding to MDA-MB-231 cells using a similar setup as in panel (D). Mean±SEM from three independent experiments is shown. Statistical difference was determined by Student’s t-test (D) or two-way ANOVA (F) method followed by Bonferoni’s multiple comparison testing. *0.05>p≥0.01; **0.01>p≥0.001; ***0.001>p. Abs, antibodies; ANOVA, analysis of variance; bsAbs, bispecific antibodies; APCP, adenosine 5′-α,β-(methylene)diphosphate; NS non-significant.