Figure 2.

CART123 exerts potent anti-leukemia and anti-endothelium activities in vitro. (A) Schematic of CAR constructs, showing that CARs consisted of anti-CD123 or anti-CD19, a CD8a hinge region, CD8 transmembrane and cytoplasmic regions, and a CD3ζ cytoplasmic region. The anti-CD123 CAR connected with EGFRt via the P2A peptide. (B) CAR expression on CART123 and CART19 was detected by biotin-conjugated goat anti-mouse IgG,F(ab’)2 fragment polyclonal antibody followed by PE-conjugated streptavidin. (C) The expression of CD123 in K562, KG-1a, MOLM-13, and NALM-6 cell lines and primary AML cells from two patients (AML-2 and AML-3) was analyzed by flow cytometry. (D) Cytotoxicity at 24 hrs of CART123, or NT, when co-cultured with MOLM-13, K562, and HUVECs was detected using 7-AAD (mean ±SEM, n=3). Data show one representative experiment. (E) Cytokine production by CART123, CART19, or NT co-cultured with primary AML cells (AML-2), myeloid leukemia cell lines K562, MOLM-13, B-ALL cell line NALM-6, and HUVECs for 24 hrs was analyzed. The culture supernatants were harvested and analyzed by ELISA (mean±SEM of triplicate). *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

Abbreviations: CAR, chimeric antigen receptor; NT, non-transduced T; HUVECs, human umbilical vein endothelial cells; 7-AAD, 7-Aminoactinomycin D; AML, acute myeloid leukemia; B-ALL, B-cell acute lymphoblastic leukemia; MFI, mean fluorescence intensity; EGFRt, truncated form of the human epidermal growth factor receptor; E:T, the effector to target cell ratio.