Pharmacologic ablation of CD44v6.CAR28z⁺ T cells coexpressing a suicide gene and rescue from hyperacute GVHD. (A) T cells were transduced with a bidirectional LV encoding for the CD44v6.CAR28z and either the thymidine kinase (TK) or the inducible caspase 9 (iC9) suicide genes (see supplemental Figure 5). At different time points after polyclonal activation and exposure to 1 μM GCV or 10 nM AP1903 (see “Material and methods”), CAR⁺ T cells were analyzed by FACS after annexin V/7AAD staining. Left: results from a representative experiment at 4 hours (annexin V^–/7AAD^–, living cells; annexin V⁺/7AAD^–, early apoptotic cells; annexin V⁺/7AAD⁺, late apoptotic cells). Right: pharmacologic ablation of CAR⁺ T cells measured as survival index (see “Material and methods,” mean ± SD from n = 3 donors). (B) The same experimental setting was used for resting, bidirectional LV-transduced T cells (mean ± SD from n = 3 donors). (C) NSG mice were infused with leukemic blasts from patient AML#4 (tumor, arrow) and, when leukemic blasts were detectable in the peripheral blood, treated with either bidirectional LV-transduced CD44v6.CAR28z⁺ or CTR.CAR28z⁺ T cells (T cells, arrow). The weight of each mouse at different time points is shown as percentage from initial. (D) Irradiated NSG mice were infused with FACS-sorted CD44v6.CAR28z⁺ T cells co-expressing iC9 (T cells, arrow) and, after losing >20% of their initial weight, received a single dose of the chemical inducer of dimerization (CID, arrow) or vehicle only. The daggers indicate death of mice from hyperacute GVHD.