CD20-BsAb enhance in vivo antitumor efficacy of CD19-CAR T cells in the Eμ-TCL1 mouse model of CLL. (A) A schematic overview of the in vivo study is presented. C57BL/6 mice (n = 10 mice per group) were transplanted with 10⁷ Eμ-TCL1 splenocyte–derived CLL cells intravenously, and disease progression was monitored through weekly blood withdrawals. After CLL establishment (median of 60% CLL cells of CD45⁺ leukocytes and >1000 CLL cells per μL in blood), the mice were conditioned with sublethal 4 Gy irradiation (day 0). Injection with 10⁶ CD19-CAR or NT T cells was performed 24 hours later (day 1). Weekly CD20-BsAb or phosphate-buffered saline (PBS) therapy was initiated the following week (day 8). Summarized frequency in blood (B) and absolute concentration (C) of CD5⁺ CD19⁺ CLL cells per mouse before and during treatment. In panel B, 3 mice reached end point criteria before the day 26 analysis (compare panel F) and the remaining mice are summarized without statistical analyses. In panel C, the symbols represent end point analysis. The number of surviving mice 60 days after therapy initiation in each treatment group are indicated on the respective graphs. The absolute concentrations are shown on pseudo-log scale to allow the plotting of mice without detectable cancer cells. (D) The Kaplan-Meier survival curves of mice treated with CD19-CAR T-cell and CD20-BsAb combination therapy, the respective monotherapies, and the NT T cells with PBS controls are shown. (E) The effects of CD20-BsAb treatment start (day 8) on total T-cell concentration are shown. The absolute T-cell concentrations in the blood of NT and CAR T-cell injected mice are depicted for day 12 (left). In addition, the endogenous and endothelial growth factor receptor⁺ CAR T-cell expansion in blood are shown separately for CAR T-cell injected mice (right). (F) CD19-CAR T-cell engraftment and expansion as monotherapy (CAR + PBS) and in combination with CD20-BsAb. The concentration of CD8⁺ CAR in blood is shown over time with relapsing mice shown in black and cured mice shown in the respective colors. (G) A second independent treatment study was performed with n = 7 CAR with PBS and n = 8 CAR with BsAb animals following the same timeline and treatment schedule as outlined in panel A. The initial response of T cells to BsAb therapy was assessed by analysis of blood and lymphoid organs 2 days after the first BsAb treatment dose (day 10) (supplemental Figure 7). Expression of CD69 (activation), PD-1 (activation/exhaustion), Ki67 (proliferation), and GrB (degranulation) in endogenous CD8⁺ and CD8⁺ CAR T cells were analyzed by flow cytometry. Exemplary histograms (left) and frequencies of marker-positive cells (right) are shown. For panel B, the 1-way analysis of variance with Tukey’s honest significant difference multiple comparison test was used. For panel D, the Mantel-Cox log-rank test was used. For panels F-G, unpaired t tests were used. In panel G, ns is not shown. ∗∗∗P ≤ .001, ∗∗P ≤ .01, ∗P ≤ .05. ns, not significant.