Evaluation of CD22 CARs in vivo. On day 0, NSG mice were injected intravenously with 5 × 105 NALM6-GL cells. On day 3 mice received 1 × 107 CAR+ T cells (HA22SH-28z (n = 5) or m971-28z (n = 5) or mock T cells (n = 5). (A) Bioluminescent imaging pre-treatment (day 3), day 7 and day 15 after intravenous injection of NALM6-GL. (B) Bioluminescent signal for each mouse over time, comparing mock, HASH-28z and m971-28z. (C) Kaplan-Meier survival curves for each group, listing significant differences between each group, survival statistics calculated using log-rank (Mantel-Cox) analysis. (D) Top row: flow cytometric analysis of transduced T cells used in the experiment, as well as CD22 and CD19 expression on NALM6-GL before injection. Bottom row: left panel, FSC versus SSC gating of splenocytes excised from a mouse on sacrifice; center-left panel, FSC versus SSC gated cells were analyzed for green fluorescence (NALM6-GL, x-axis) and CD45-PerCp (y-axis); center right, CD45-gated cells were stained for CD3-APC (y-axis) and for CAR expression; right panel, GFP+ gated cells were stained for CD22 and CD19. (E) In a separate experiment, carried out identically, mice were killed on day 12, and analyzed for CAR expression on gated human T cells in the bone marrow, spleen, and blood. Percentage of T cells expressing CARs is plotted (y-axis), and significant differences, using unpaired 2-tailed t test, are shown.
