Figure 5.

Mature human T-cells expressing a third-generation chimeric antigen receptor (CAR) against CD123, or CD123 CAR and inducible caspase 9 (iCasp9) in combination are functional in vitro. (a) Schematic figure of the MPSV.CD123 CAR construct. Self-inactivating (SIN) alpharetroviral vectors encoding for a third-generation CAR against the human surface molecule CD123 were generated. (b) Human peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 and anti-CD28 antibodies for 2 days, transduced on 2 consecutive days, and expanded for 4 more days before being used in a cytotoxicity assay. TdTomato.CD123-transduced 293T cells were used as target cells. Decreasing numbers of untransduced or CAR-transduced PBMCs were cultured with target cells in triplicates for 2 days. TdTomato expression was used as a marker for surviving target cells in immunofluorescence microscopy studies. (c) Cytotoxicity of the transduced effectors was determined by means of flow cytometry. Cell suspensions containing CAR-transduced effector cells and targets (either 293T cells only or 293T cells expressing CD123) at indicated ratios were stained for CD3. The percentage of viable CD3^neg cells (surviving target cells) after incubation was used as an indirect parameter for cytotoxicity (left panel). Using an IFNγ release assay, T-cells (2 × 10⁵) and target cells (2 × 10⁴) were incubated at fixed effector:target ratios of 10:1 in 96-well plates. After 24 hours, supernatant was harvested and used in duplicates for an IFNγ enzyme-linked immunosorbent assay (right panel) (n = 2). Untransduced (untd) T-cells were used as control. (d) Schematic figure of the MPSV.CD123 CAR.iCasp9 vector. Human PBMCs were transduced with this bicistronic construct. Transgene positive cells with a functional CAR were identified by specific cell binding of a His Tag-labeled CD123 peptide. Transduced cells were incubated with the dimerizer for 48 hours. CAR.GFP-transduced PBMCs were used as control (n = 3). Apoptosis was quantified by staining viable transgene positive (CAR+ or GFP+) Annexin V-PBMCs. Indicated are unique 5 (U5), repeat (R), and SIN unique 3 (ΔU3) regions, long terminal repeat (LTR), packaging signal (ψ), myeloproliferative sarcoma virus promoter (MPSV), single chain variable fragment (scFV), internal ribosomal entry site (IRES), woodchuck posttranscriptional regulatory element (PRE) and direct repeat element (DR).