Figure 4.

CD19/CD20 bi-specific constructs are highly expressed and confer chimeric antigen receptor (CAR) T cells cytotoxicity and cytokine production. (A) Schematic of bi-specific single CAR constructs targeting CD19 and CD20 in a tandem (Tan) configuration. Histograms showing CAR expression. Quantitative analysis of CAR expression (Protein L staining) at day 7 post-transduction compared with 19-28z cells. (B) Real-time cytotoxicity assay (xCelligence) was performed with the different bi-specific CAR T cells and single 19-28z against Raji-CD19^High, Raji-CD19^Low and Raji-CD19^KO target cells at 1:1 E:T ratio. (C) Concentration of interferon gamma (IFN-γ), interleukin (IL)-2, IL-6 and tumor necrosis factor alpha (TNF-α) measured by ELLA in supernatants from co-cultures of bi-specific CAR T cells and single 19-28z with Raji-CD19^-High, Raji-CD19^Low and Raji-CD19^KO after 24 hours compared with untransduced T (UT) cells. All conditions were normalized to the lowest CAR expression using UT cells, reaching same number of CAR T cells and total T cells per group. Representative results of three independent experiments are shown. Data are shown as mean±SD. NS, not significant; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. One-way analysis of variance (ANOVA) was performed with Dunnet’s multiple comparison test against UT.