Fig. 6. Functional applications of combYSelect heterodimers.

(A) Representation of the cell-bridging assay, in which the Fc of protomer A (combYSelect1 and combYSelect2) is engineered with an anti-CD20 rituximab Fab and the Fc protomer B (combYSelect1) or protomer C (combYSelect2) is attached to an anti-HER2 nanobody. Binding to CFSE-stained Raji cells and Calcein-Violet BT474 cells will be assessed. (B) Flow cytometry density plots and a scatterplot depicting cell cluster formations of Raji and BT474 cells when combYSelect1, combYSelect2, a nonspecific IgG1 isotype control, or no antibody was added to the cell mixture. Statistical significance (n = 5) was determined by one-way ANOVA with Tukey’s multiple-comparison test (ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001). Each combYSelect IgG was compared to both the no antibody and isotype control. (C) Depiction of combYSelect2 cytokine trap and the luciferase inhibition cell-based assay. (D) Percentage of luciferase response, normalized to that when no inhibitor is added, is plotted as a function of varying inhibitor concentration in the presence of 5 pM IL-1β. The IC₅₀ values were determined using a nonlinear least square fit.