Fig. 3.

In vitro comparison of premixed and consecutive MATCH. (A) Raji B-cell depletion after 24 h incubation with either premixed αCD20 MATCH conjugates (left) or consecutive αCD20 MATCH (right). The T cell-to-Raji cell ratio was 1:3. Depletion of Raji cells and T-cell PD-1 expression was measured by flow cytometry (FACSCanto). (B) Two-dimensional dose heat maps were generated using luciferase-expressing Raji cells co-cultured with T-cells in a 1:1 ratio for 24 h. Raji cell death was quantified by luciferin addition and subsequent bioluminescence imaging (bioluminescence indicating residual target cells). (C) In vitro target cell death for titrations of each MATCH formulation is compared to blinatumomab (line graph) and example residual cell histograms are presented for 5 nM treatments. (D) In vitro T-cell cytokine release comparison of αCD19 and αCD20 MATCH to blinatumomab was performed using a T/NK cell cytokine multiplex panel, with 5 nM dose presented here. Additional data can be found in Supplementary Figs. S5–S7. (E) Competitive binding assays were performed by flow cytometry on Fab′_CD3-MORF2, Fab′_CD19-MORF1, and blinatumomab for comparison. (F) Premixed αCD20 MATCH target cell depletion in combination with an αIL-10 antibody. (G) Premixed αCD20 MATCH target cell depletion in combination with an αPD-1 antibody. All experiments were performed in triplicate. ***p < 0.001, **p < 0.01, *p < 0.05, n.s. not significant by One-Way ANOVA and Tukey test.