Lentiviral transduction, expansion, and selection of bi-specific T cells. (A) Scheme for simultaneous virus-specific stimulation and CD19-CAR transduction. T_CM cells were plated at 10⁶ cells/well into 24-well plates with 10⁶ peptide-pulsed γ-irradiated PBMCs/well and IL-2 (50 IU/mL), and transduced on day 1 with CD19-CAR lentivirus supernatant. (B) Staining of transduced and untransduced T cells with HLA tetramer, anti-Fc, and anti-EGFR reagents after 10 days of culture. Data are shown for a culture stimulated with an HLA-B*0702_CMVpp65 peptide-pulsed PBMCs. Similar data were obtained for T_CM cells stimulated with A*0101_CMVpp50, A*0201_CMVpp65, A*0201_EBV-BMLF1, and B*0801_EBV-BZLF1 peptides. (C) Growth of bi-specific T cells after stimulation with CD19⁺ EBV-transformed B cells (TM-LCL) at various T-cell:LCL ratios during a 10-day stimulation cycle. Data are pooled from 5 independent experiments and the mean fold expansion in cell number and SEM are shown. (D) CD19-CAR⁺ and tetramer-positive T cells are enriched over 2 cycles of stimulation with CD19⁺ TM-LCL. The bi-specific T cells were stimulated with a 1:7 ratio of TM-LCL, fed with 50 IU/mL IL-2 and the frequency of cells that bound the HLA tetramer and expressed cell-surface EGFR was determined on day 10-13 (left panel, CAR⁺ cells; right panel, tetramer-positive cells). Data are pooled from 5 independent experiments (mean and SEM) with 4 viral epitopes (A*0101_CMVpp50, A*0201_CMVpp65, B*0702_CMVpp65, and A*0201_EBV-BMLF1). (E) Purification of tetramer positive bi-specific cells with reversible class I MHC streptamer. Streptamer selection was performed after the first stimulation with CD19⁺ TM-LCLs. Shown are the data for selection of HLA B*0702_CMVpp65 bi-specific T cells, which is representative of 4 independent experiments with A*0101_CMVpp50, A*0201_CMVpp65, B*0702_CMVpp65, and A*0201_EBV-BMLF1 streptamers. The yield was a median of 26.9% (range, 15.0%-43.8%). (F) Purity of final bi-specific T_CM-derived cell product. After MHC streptamer selection, a second stimulation with TM-LCL was performed and the bi-specific T cells were stained with the class I HLA tetramer and with anti-EGFR on day 10 after stimulation. Data are shown for a bi-specific (B*0702_CMVpp65; CD19CAR) T-cell product and are representative of 4 independent experiments.