Fig. 2. A single dose of CD33-CAR-NK cells displays potent anti-tumor efficacy in OCI-AML2 engrafted NSG-SGM3 mice.
A Scheme of the in vivo evaluation of a single treatment with CD33-CAR-NK cells (1 × 107 intravenously) followed by subcutaneous treatment with IL-2 in OCI-AML2 (Luc+) xenograft NSG-SGM3 mice. B Total flux analysis as well as representative BLI images of differently treated OCI-AML2 (Luc+) engrafted NSG-SGM3 mice over time (d7 n = 7; d14 n = 6; d21 n = 5 per group). Mice received a single dose of 1 × 107 NK cells day 3 post AML cell injection. At day 21, 4 out of 5 mice (80%) that were treated with CD33-CAR-NK cells show severely reduced leukemic burden compared to untreated mice (UT) or mice which received untransduced (UTD)-NK cells. C Serum analysis of blood day 3 before AML injection and day 1 post first NK cell application shows significantly increased levels of GM-CSF as well as INF-γ for mice that had received CD33-CAR-NK cells (n = 3). Mean ± SD. D Total flux analysis of femurs/tibiae and spleens, as well as flow cytometry analysis of isolated cells from BMs or spleens at day 7, 14, and 21 post tumor cell injection, revealed the absence of GFP-positive tumor cells in CD33-CAR-NK-treated mice as well as increased NK cell infiltration (day 7/14 n = 1; day 21 n = 2 per group). Values of zero were set to 1 for total flux analysis. Median ± range. Flow cytometry-based CAR expression analysis of BM- (E) or spleen- (F) infiltrating NK cells at day 14 and 21 revealed the presence of mainly CAR-positive cells (day 14 n = 1; day 21 n = 2 per group). Mean ± SD. G Confocal microscopy imaging shows GFP-positive leukemia cells in BM of UTD-NK treated NSG-SGM3 mice at day 21 while absent in mice that received CD33-CAR-NK cells. Images from one representative animal are shown. Statistical analysis was performed by Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).